Antibodies to il-6 and their uses

ABSTRACT

Antibodies and antigen-binding portions thereof that bind to human IL-6 are provided. Also provided are nucleic acids encoding such antibodies and antigen binding portions, methods of making such antibodies and antigen binding portions, compositions comprising such antibodies or antigen binding portions, and uses of such antibodies or antigen binding portions.

CROSS-REFERENCE TO RELATED PATENTS AND PATENT APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/857,867, filed Apr. 5, 2013, now U.S. Pat. No.8,846,037; which is a divisional application of U.S. patent applicationSer. No. 13/454,296, filed Apr. 24, 2012, now U.S. Pat. No. 8,436,158;which is a divisional application of U.S. patent application Ser. No.12/480,991, filed Jun. 9, 2009, now U.S. Pat. No. 8,188,235, whichclaims the benefit of U.S. Provisional Patent Application Ser. No.61/073,430 filed Jun. 18, 2008. The disclosures of all of theaforementioned applications are incorporated herein by reference intheir entirety.

The instant application contains a Sequence Listing that has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII file, created on Aug. 29, 2014, isnamed PCFC-596-104-Sequence-Listing.txt and is 50,115 bytes in size.

JOINT RESEARCH AGREEMENT

The disclosure and claims herein were made as a result of activitiesundertaken within the scope of a joint research agreement in effect onor before the date the claimed invention was made between Pfizer Inc.and Medarex, Inc.

BACKGROUND

This invention relates to antibodies and antigen-binding portionsthereof that bind to human IL-6. This invention also relates to nucleicacids encoding such antibodies and antigen binding portions thereof;methods of making such antibodies and antigen binding portions thereof;compositions comprising such antibodies or antigen binding portionsthereof; and uses of such antibodies or antigen binding portionsthereof.

Interleukin-6 (IL-6), which is also known as interferon B2 (IFNB2), is apleiotropic cytokine that belongs to the family of gp130 ligands and isproduced by many cell types, including T lymphocytes, fibroblasts andmonocytes. IL-6 is produced constitutively at low levels and is readilyinduced by infectious stimuli or inflammatory cytokines. IL-6 binds to aspecific receptor IL-6R (gp80) which heterodimerizes with cell-bound orsoluble gp130 to form a functional receptor complex. Binding of IL-6 toits receptor initiates cellular events including activation of theJAK-STAT3 pathway and ras-mediated MAP kinase signaling. IL-6 can elicita diverse array of effects such as proliferation and differentiation ofB cells and monocytes, T cell activation, hematopoiesis, osteoclastactivation, keratinocyte growth, neuronal growth, hepatocyte activationand acute phase protein induction from hepatocytes.

IL-6 plays an important role in B cell abnormalities as demonstrated insystemic lupus erythematosus, multiple myeloma and lymphoproliferativedisorders. Similarly, IL-6 is also implicated in the pathogenesis ofautoimmune and inflammatory diseases such as rheumatoid arthritis andosteoarthritis. Recently, indirect evidence suggests an associationbetween IL-6 and chronic obstructive pulmonary disease and insulinresistance in type 2 diabetes. IL-6 has both pro-inflammatory andanti-inflammatory effects in the immune system, indicating that thiscytokine likely plays a central role in regulating the physiologicalresponse to disease. Therefore, targeting IL-6 can potentially providetherapeutic benefit in a variety of disease areas.

An increase in the production of IL-6 has been observed in a number ofdiseases including: Alzheimer's disease, autoimmune diseases, such asrheumatoid arthritis, inflammation, myocardial infarction, Paget'sdisease, osteoporosis, liver fibrosis, solid tumors (renal cellcarcinoma), prostatic and bladder cancers, neurological cancers, andB-cell malignancies (e.g., Casteleman's disease, certain lymphomas,chronic lymphocytic leukemia, and multiple myeloma). Research hasindicated that IL-6 is linked to the pathogenesis of many of thesediseases, particularly, cancer and rheumatoid arthritis and, therefore,blocking IL-6 should translate into clinical benefits.

SUMMARY

An isolated antibody or antigen-binding portion thereof thatspecifically binds IL-6 and may act as an IL-6 receptor antagonist, andcompositions comprising the antibody or portion are produced.

Compositions comprising (i) the heavy and/or light chain, the variabledomains thereof, or antigen-binding portions thereof, of the anti-IL-6antibody, or nucleic acid molecules encoding them; and (ii) apharmaceutically acceptable carrier are provided. The compositions mayfurther comprise another component, such as a therapeutic agent or adiagnostic agent.

Diagnostic and therapeutic methods are also provided. Similarly, theanti-IL-6 antibodies and antigen-binding portions thereof are providedfor the manufacture of medicaments to treat inflammatory andnon-inflammatory disorders.

Vectors and host cells comprising the nucleic acid molecules, as well asmethods of recombinantly producing the polypeptides encoded by thenucleic acid molecules are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F show an alignment of the germline amino acid sequences ofthe heavy and light chain variable regions compared to the respectiveanti-IL-6 antibodies 9C8, 9C8 N68T T83S and 22B5 heavy and light chainvariable regions (only mismatches are shown for the 9C8, 9C8 N68T T83S,and 22B5 antibody). FIG. 1A shows the heavy chain variable region of the22B5 antibody. FIG. 1B shows the light chain variable region of the 22B5antibody. FIG. 1C shows the heavy chain variable region of the 9C8antibody. FIG. 1D shows the light chain variable region of the 9C8antibody. FIG. 1E shows the heavy chain variable region of the 9C8 N68TT83S antibody. FIG. 1F shows the light chain variable region of the 9C8N68T T83S antibody. The CDRs are underlined and mismatched gap(s) areindicated by a pound sign (#).

FIG. 2 shows the total serum C-reactive protein (CRP) for the vehicleand the 9C8 N68T T83S IgG₂ antibody as determined byElectrochemiluminescence Immuno-Assay. Each point represents an averagevalue of serum CRP from 3 cynomolgus monkeys (±SE) dosed with vehicle oranti-IL-6 antibody 9C8 N68T T83S IgG₂ at 0.5 mg/kg and 5.0 mg/kg. SerumCRP was measured by Meso Scale Discovery (MSD). LPS was administered atthe 0 hour time point.

DETAILED DESCRIPTION Definitions and General Techniques

The term “antibody” is synonymous with immunoglobulin and is to beunderstood as commonly known in the art. In particular, the termantibody is not limited by any particular method of producing theantibody. For example, the term antibody includes, inter alia,recombinant antibodies, monoclonal antibodies, and polyclonalantibodies.

The basic antibody structural unit is a tetramer. Each tetramer iscomposed of two identical pairs of polypeptide chains, each pair havingone “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 120 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa and lambda light chains. Heavy chains areclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 3 or more amino acids.

The variable regions of each heavy/light chain pair (V_(H) and V_(L)),respectively, form the antigen binding site. Thus, an intact IgGantibody, for example, has two binding sites. Except in bifunctional orbispecific antibodies, the two binding sites are the same.

The variable regions of the heavy and light chains exhibit the samegeneral structure of relatively conserved framework regions (FR) joinedby three hyper variable regions, also called complementary determiningregions or CDRs. The term “variable” refers to the fact that certainportions of the variable domains differ extensively in sequence amongantibodies and are used in the binding and specificity of eachparticular antibody for its particular antigen. The variability,however, is not evenly distributed throughout the variable domains ofantibodies, but is concentrated in the CDRs, which are separated by themore highly conserved FRs. The CDRs from the two chains of each pair arealigned by the FRs, enabling binding to a specific epitope. FromN-terminal to C-terminal, both light and heavy chains comprise thedomains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of aminoacids to each domain is in accordance with the definitions of KabatSequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol.196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989), thedisclosures of which are herein incorporated by reference.

As used herein, an antibody that is referred to by number is the same asa monoclonal antibody that is obtained from the hybridoma of the samenumber. For example, anti-IL-6 monoclonal antibody 9C8 is the sameantibody as one obtained from hybridoma 9C8, or a subclone thereof.

The term “analog” or “polypeptide analog” means a polypeptide thatcomprises a segment that has substantial identity to some referenceamino acid sequence and has substantially the same function or activityas the reference amino acid sequence. Typically, polypeptide analogscomprise one or more conservative amino acid substitution (or insertionor deletion) with respect to the reference sequence. Analogs can be atleast 20 or 25 amino acids long, or can be at least 50, 60, 70, 80, 90,100, 150 or 200 amino acids long or longer, and can often be as long asthe full-length polypeptide. Some embodiments include polypeptideanalogs with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17substitutions from the reference amino acid sequence. In some instances,the reference amino acid sequence is a germline sequence. Analogs ofantibodies or immunoglobulin molecules can be readily prepared by thoseof ordinary skill in the art.

As discussed herein, amino acid substitutions to an IL-6 antibody orantigen-binding portion thereof are those which typically: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) delete orcreate a site for glycosylation, or (4) confer or modify otherphysicochemical or functional properties of such analogs, but stillretain specific binding to IL-6.

Analogs can include various substitutions to the normally-occurringpeptide sequence. For example, single or multiple amino acidsubstitutions, preferably conservative amino acid substitutions, may bemade in the normally-occurring sequence. A conservative amino acidsubstitution typically does not substantially change the structuralcharacteristics of the parent sequence.

The term “antigen-binding portion” of an antibody refers to a fragmentof an antibody that retains the ability to specifically bind to anantigen (e.g., IL-6). It has been shown that the antigen-bindingfunction of an antibody can be performed by fragments of a full-lengthantibody. Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include: (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H)1domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and C_(H)1 domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody; (v) a domain antibody, (dAb) (Ward et al., (1989) Nature341:544-546), which consists of a V_(H) domain; and (vi) an isolatedcomplementary determining region (CDR). Furthermore, although the twodomains of the Fv fragment, V_(L) and V_(H), are coded for by separategenes, they can be joined, using recombinant methods, by a syntheticlinker that enables them to be made as a single protein chain in whichthe V_(L) and V_(H) regions pair to form monovalent molecules (known assingle chain Fv (scFv)). Such single chain antibodies are also intendedto be encompassed within the term “antigen-binding portion” of anantibody. Other forms of single chain antibodies, such as diabodies arealso encompassed. Diabodies are bivalent, bispecific antibodies in whichV_(H) and V_(L) domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites. In addition, one or more CDRs from an antibody may beincorporated into a larger polypeptide chain, which can be covalently ornon-covalently linked to another. In embodiments having one or morebinding sites, the binding sites may be identical to one another or maybe different.

Still further, an antibody or antigen-binding portion thereof may bepart of larger immunoadhesion molecules, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule and use of a cysteine residue, a marker peptideand a C-terminal polyhistidine tag to make bivalent and biotinylatedscFv molecules. Other examples include where one or more CDRs from anantibody are incorporated into a molecule either covalently ornoncovalently to make it an immunoadhesin that specifically binds to anantigen of interest, such as IL-6. In such embodiments, the CDR(s) maybe incorporated as part of a larger polypeptide chain, may be covalentlylinked to another polypeptide chain, or may be incorporatednoncovalently. Antibody portions, such as Fab and F(ab′)₂ fragments, canbe prepared from whole antibodies using any suitable technique, such aspapain or pepsin digestion, respectively, of whole antibodies. Moreover,antibodies, antibody portions and immunoadhesion molecules can beobtained using various recombinant DNA techniques.

The term “chimeric antibody” means an antibody that comprises regionsfrom two or more different antibodies, including antibodies fromdifferent species. For example, one or more of the CDRs of a chimericantibody can be derived from a human IL-6 antibody. In one example, theCDRs from a human antibody can be combined with CDRs from a non-humanantibody, such as mouse or rat. In another example, all of the CDRs canbe derived from human IL-6 antibodies. In another example, the CDRs frommore than one human IL-6 antibody can be combined in a chimericantibody. For instance, a chimeric antibody may comprise a CDR1 from thelight chain of a first human IL-6 antibody, a CDR2 from the light chainof a second human IL-6 antibody and a CDR3 from the light chain of athird human IL-6 antibody, and CDRs from the heavy chain may be derivedfrom one or more other IL-6 antibodies. Further, the framework regionsmay be derived from one of the IL-6 antibodies from which one or more ofthe CDRs are taken or from one or more different human antibodies.Further, the term “chimeric antibody” is intended to encompass any ofthe above mentioned combinations where the combinations involved humanand non-human antibodies.

The term “compete” means that a first antibody, or an antigen-bindingportion thereof, competes for binding with a second antibody, or anantigen-binding portion thereof, where binding of the first antibodywith its cognate epitope is detectably decreased in the presence of thesecond antibody compared to the binding of the first antibody in theabsence of the second antibody. The alternative, where the binding ofthe second antibody to its epitope is also detectably decreased in thepresence of the first antibody, can, but need not be the case. That is,a first antibody can inhibit the binding of a second antibody to itsepitope without that second antibody inhibiting the binding of the firstantibody to its respective epitope. However, where each antibodydetectably inhibits the binding of the other antibody with its cognateepitope or ligand, whether to the same, greater, or lesser extent, theantibodies are said to “cross-compete” with each other for binding oftheir respective epitope(s). Regardless of the mechanism by which suchcompetition or cross-competition occurs (e.g., steric hindrance,conformational change, or binding to a common epitope, or portionthereof), the skilled artisan would appreciate, based upon the teachingsprovided herein, that such competing and/or cross-competing antibodiescan be useful for the methods disclosed herein.

The term “conservative amino acid substitution” means an amino acidresidue is substituted by another amino acid residue having a side chainR group with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well-known to thoseof skill in the art. Examples of groups of amino acids that have sidechains with similar chemical properties include 1) aliphatic sidechains: glycine, alanine, valine, leucine, and isoleucine; 2)aliphatic-hydroxyl side chains: serine and threonine; 3)amide-containing side chains: asparagine and glutamine; 4) aromatic sidechains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains:lysine, arginine, and histidine; 6) acidic side chains: aspartic acidand glutamic acid; and 7) sulfur-containing side chains: cysteine andmethionine. Conservative amino acids substitution groups can be, forexample, valine-leucine-isoleucine, phenylalanine-tyrosine,lysine-arginine, alanine-valine, glutamate-aspartate, andasparagine-glutamine.

A conservative replacement is also any change having a positive value inthe PAM250 log-likelihood matrix disclosed in Gonnet et al., Science256:1443-45 (1992), incorporated herein by reference. A “moderatelyconservative” replacement is any change having a non-negative value inthe PAM250 log-likelihood matrix.

“Contacting” refers to bringing an antibody or antigen binding portionthereof and a target IL-6, or epitope thereof, together in such a mannerthat the antibody can affect the biological activity of the IL-6. Such“contacting” can be accomplished in vitro, e.g., in a test tube, a petridish, or the like. In a test tube, contacting may involve only anantibody or antigen binding portion thereof and IL-6 or epitope thereofor it may involve whole cells. Cells may also be maintained or grown incell culture dishes and contacted with antibodies or antigen bindingportions thereof in that environment. In this context, the ability of aparticular antibody or antigen binding portion thereof to affect an IL-6related disorder, i.e., the IC₅₀ of the antibody, can be determinedbefore use of the antibody in vivo with more complex living organisms.For cells outside the organism, multiple methods exist, and arewell-known to those skilled in the art, to contact IL-6 with theantibodies or antigen-binding portions thereof.

The term “ELISA” refers to an enzyme-linked immunosorbent assay. Thiskind of assay is well known to those of skill in the art.

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin or T-cell receptor or otherwise interactingwith a molecule. Epitopic determinants generally consist of chemicallyactive surface groupings of molecules such as amino acids orcarbohydrate or sugar side chains and generally have specific threedimensional structural characteristics, as well as specific chargecharacteristics. An epitope may be “linear” or “conformational.” In alinear epitope, all of the points of interaction between the protein andthe interacting molecule (such as an antibody) occur linearly along theprimary amino acid sequence of the protein. In a conformational epitope,the points of interaction occur across amino acid residues on theprotein that are separated from one another. Once a desired epitope onan antigen is determined, antibodies to that epitope can be generated.An approach to achieve this is to conduct cross-competition studies tofind antibodies that competitively bind with one another, i.e., theantibodies compete for binding to the antigen. A high throughput processfor “binning” antibodies based upon their cross-competition is describedin International Patent Publication No. WO 03/48731.

The term “expression control sequence” as used herein meanspolynucleotide sequences that are necessary to effect the expression andprocessing of coding sequences to which they are ligated. Expressioncontrol sequences include appropriate transcription initiation,termination, promoter and enhancer sequences; efficient RNA processingsignals such as splicing and polyadenylation signals; sequences thatstabilize cytoplasmic mRNA; sequences that enhance translationefficiency (i.e., Kozak consensus sequence); sequences that enhanceprotein stability; and when desired, sequences that enhance proteinsecretion. The nature of such control sequences differs depending uponthe host organism; in prokaryotes, such control sequences generallyinclude promoter, ribosomal binding site, and transcription terminationsequence; in eukaryotes, generally, such control sequences includepromoters and transcription termination sequence. The term “controlsequences” is intended to include, at a minimum, all components whosepresence is essential for expression and processing, and can alsoinclude additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences.

The term “germline” refers to the nucleotide sequences of the antibodygenes and gene segments as they are passed from parents to offspring viathe germ cells. This germline sequence is distinguished from thenucleotide sequences encoding antibodies in mature B cells which havebeen altered by recombination and hypermutation events during the courseof B cell maturation.

The term “human antibody” means any antibody in which the variable andconstant domain sequences are human sequences. The term encompassesantibodies with sequences derived from human genes, including thosewhich have been changed, e.g., to decrease possible immunogenicity,increase affinity, eliminate cysteine residues that might causeundesirable folding, etc. The term also encompasses such antibodiesproduced recombinantly in non-human cells, which might impartglycosylation not typical of human cells. These antibodies may beprepared in a variety of ways.

The term “humanized antibody” refers to antibodies of non-human origin,wherein the amino acid residues that are characteristic of antibodysequences of the non-human species are replaced with residues found inthe corresponding positions of human antibodies. This “humanization”process can reduce the immunogenicity in humans of the resultingantibody. Antibodies of non-human origin can be humanized using anysuitable technique well known in the art. The antibody of interest maybe engineered by recombinant DNA techniques to substitute the CH1, CH2,CH3, hinge domains, and/or the framework domain with the correspondinghuman sequence. The term “humanized antibody” further includes withinits meaning, chimeric human antibodies and CDR-grafted antibodies.Chimeric human antibodies include the V_(H) and V_(L) of an antibody ofa non-human species and the C_(H) and C_(L) domains of a human antibody.The CDR-transplanted antibodies result from the replacement of CDRs ofthe V_(H) and V_(L) of a human antibody with those of the V_(H) andV_(L), respectively, of an antibody of an animal other than a human.

The term “isolated polynucleotide” as used herein means a polynucleotideof genomic, cDNA, or synthetic origin or a combination thereof, which byvirtue of its origin the “isolated polynucleotide” (1) is not associatedwith all or a portion of polynucleotides with which the “isolatedpolynucleotide” is found in nature, (2) is operably linked to apolynucleotide to which it is not linked in nature, or (3) does notoccur in nature as part of a larger sequence.

The term “isolated protein”, “isolated polypeptide” or “isolatedantibody” is a protein, polypeptide or antibody that by virtue of itsorigin or source of derivation: (1) is not associated with naturallyassociated components that accompany it in its native state; (2) is freeof other proteins from the same species; (3) is expressed by a cell froma different species; or (4) does not occur in nature. Thus, apolypeptide that is, e.g., chemically synthesized or synthesized in acellular system different from the cell from which it naturallyoriginates will be “isolated” from its naturally associated components.A protein may also be rendered substantially free of naturallyassociated components by isolation, using any suitable proteinpurification technique.

Examples of isolated antibodies include an IL-6 antibody that has beenaffinity purified using IL-6, and an IL-6 antibody that has beensynthesized by a cell line in vitro.

The term “K_(D)” refers to the binding affinity equilibrium constant ofa particular antibody-antigen interaction. An antibody is said tospecifically bind an antigen when the K_(D) is ≦1 mM, preferably ≦100nM, and most preferably ≦10 nM. A K_(D) binding affinity constant can bemeasured by surface plasmon resonance, for example using the BIACORE™system as discussed in EXAMPLE 7.

The term “k_(off)” refers to the dissociation rate constant of aparticular antibody-antigen interaction. A k_(off) dissociation rateconstant can be measured by surface plasmon resonance, for example usingthe BIACORE™ system as discussed in EXAMPLE 7.

The term “naturally occurring nucleotides” as used herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” as used herein includes, for example, nucleotides withmodified or substituted sugar groups. The term “oligonucleotidelinkages” referred to herein includes oligonucleotide linkages such as,for example, phosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoroamidate. An oligonucleotide can include a label for detection,if desired.

“Operably linked” sequences include both expression control sequencesthat are contiguous with the gene of interest and expression controlsequences that act in trans or at a distance to control the gene ofinterest.

The term “percent sequence identity” in the context of nucleic acidsequences means the residues in two sequences that are the same whenaligned for maximum correspondence. The length of sequence identitycomparison may be over at least about nine nucleotides, usually at leastabout 18 nucleotides, more usually at least about 24 nucleotides,typically at least about 28 nucleotides, more typically at least about32 nucleotides, and at least about 36, 48 or more nucleotides. There area number of different algorithms known in the art which can be used tomeasure nucleotide sequence identity. For instance, polynucleotidesequences can be compared using FASTA, Gap or Besffit, which areprograms in Wisconsin Package Version 10.0, Genetics Computer Group(GCG), Madison, Wis. FASTA, which includes, e.g., the programs FASTA2and FASTA3, provides alignments and percent sequence identity of theregions of the best overlap between the query and search sequences(Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol.132:185-219 (2000); Pearson, Methods Enzymol. 266:227-258 (1996);Pearson, J. Mol. Biol. 276:71-84 (1998); incorporated herein byreference). Default parameters for a particular program or algorithm aretypically used. For instance, percent sequence identity between nucleicacid sequences can be determined using FASTA with its default parameters(a word size of 6 and the NOPAM factor for the scoring matrix) or usingGap with its default parameters as provided in GCG Version 6.1,incorporated herein by reference.

A reference to a nucleotide sequence encompasses its complement unlessotherwise specified. Thus, a reference to a nucleic acid having aparticular sequence should be understood to encompass its complementarystrand, with its complementary sequence.

The term “percent sequence identity” in the context of amino acidsequences means the residues in two sequences that are the same whenaligned for maximum correspondence. The length of sequence identitycomparison may be over at least about five amino acids, usually at leastabout 20 amino acids, more usually at least about 30 amino acids,typically at least about 50 amino acids, more typically at least about100 amino acids, and even more typically about 150, 200 or more aminoacids. There are a number of different algorithms known in the art thatcan be used to measure amino acid sequence identity. For instance, aminoacid sequences can be compared using FASTA, Gap or Bestfit, which areprograms in Wisconsin Package Version 10.0, Genetics Computer Group(GCG), Madison, Wis.

Sequence identity for polypeptides is typically measured using sequenceanalysis software. Protein analysis software matches sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG contains programs such as “Gap” and “Bestfit” whichcan be used with default parameters as specified by the programs todetermine sequence homology or sequence identity between closely relatedpolypeptides, such as homologous polypeptides from different species oforganisms or between a wild type protein and an analog thereof. See,e.g., GCG Version 6.1 (University of Wisconsin, Wis.). Polypeptidesequences also can be compared using FASTA using default or recommendedparameters, see GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3)provides alignments and percent sequence identity of the regions of thebest overlap between the query and search sequences (Pearson, MethodsEnzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219(2000)). Another algorithm when comparing a sequence to a databasecontaining a large number of sequences from different organisms is thecomputer program BLAST, especially blastp or tblastn, using defaultparameters as supplied with the programs. See, e.g., Altschul et al., J.Mol. Biol. 215:403-410 (1990); Altschul et al., Nucleic Acids Res.25:3389-402 (1997).

The term “recombinant host cell” (or simply “host cell”), as usedherein, means a cell into which a recombinant expression vector has beenintroduced. It should be understood that “recombinant host cell” and“host cell” mean not only the particular subject cell but also theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

A protein or polypeptide is “substantially pure,” “substantiallyhomogeneous,” or “substantially purified” when at least about 60 to 75%of a sample exhibits a single species of polypeptide. The polypeptide orprotein may be monomeric or multimeric. A substantially pure polypeptideor protein can typically comprise about 50%, 60%, 70%, 80% or 90% w/w ofa protein sample, more usually about 95%, and preferably can be over 99%pure. Protein purity or homogeneity may be indicated by any suitablemeans, such as polyacrylamide gel electrophoresis of a protein samplefollowed by visualizing a single polypeptide band upon staining the gelwith a stain. As one skilled in the art will appreciate, higherresolution may be provided by using HPLC or other means forpurification.

The term “substantial similarity” or “substantial sequence similarity,”when referring to a nucleic acid or fragment thereof, means that whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 85%, at least about 90%,and at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, asmeasured by any well-known algorithm of sequence identity, such asFASTA, BLAST or Gap, as discussed above.

As applied to polypeptides, the term “substantial identity” or“substantial similarity” means that two amino acid sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap weights as supplied with the programs, share at least 70%, 75% or80% sequence identity, preferably at least 90% or 95% sequence identity,and more preferably at least 97%, 98% or 99% sequence identity. Incertain embodiments, residue positions that are not identical differ byconservative amino acid substitutions.

The term “surface plasmon resonance” refers to an optical phenomenonthat allows for the analysis of real-time biospecific interactions bydetection of alterations in protein concentrations within a biosensormatrix, for example using the BIACORE™ system (Pharmacia Biosensor AB,Uppsala, Sweden and Piscataway, N.J.). For further descriptions, seeJonsson U. et al., Ann. Biol. Clin. 51:19-26 (1993); Jonsson U. et al.,Biotechniques 11:620-627 (1991); Jonsson B. et al., J. Mol. Recognit.8:125-131 (1995); and Johnsson B. et al., Anal. Biochem. 198:268-277(1991).

“Therapeutically effective amount” refers to that amount of thetherapeutic agent being administered which will relieve to some extentone or more of the symptoms of the disorder being treated. In referenceto the treatment of rheumatoid arthritis, a therapeutically effectiveamount refers to that amount which has at least one of the followingeffects: reducing the structural damage of joints; inhibiting (that is,slowing to some extent, preferably stopping) the accumulation of fluidin the joint area; and relieving to some extent (or, preferably,eliminating) one or more symptoms associated with rheumatoid arthritis.

“Treat”, “treating” and “treatment” refer to a method of alleviating orabrogating a biological disorder and/or its attendant symptoms.

The term “utilizes” with reference to a particular gene means that theamino acid sequence of a particular region in an antibody was ultimatelyderived from that gene during B-cell maturation. For example, the phrase“a heavy chain variable region amino acid sequence that utilizes a humanV_(H)-3 family gene” refers to the situation where the V_(H) region ofthe antibody was derived from the VH-3 family of gene segments duringB-cell maturation. In human B-cells, there are more than 30 distinctfunctional heavy chain variable genes with which to generate antibodies.Use of a particular heavy chain variable gene, therefore, is indicativeof a binding motif of the antibody-antigen interaction with respect tothe combined properties of binding to the antigen and functionalactivity. As will be appreciated, gene utilization analysis providesonly a limited overview of antibody structure. As human B-cellsstochastically generate V-D-J heavy or V-J kappa light chaintranscripts, there are a number of secondary processes that occur,including, without limitation, somatic hypermutation, additions, andCDR3 extensions. See, for example, Mendez et al. Nature Genetics15:146-156 (1997).

The term “vector”, as used herein, means a nucleic acid molecule capableof transporting another nucleic acid to which it has been linked. Insome cases, the vector is a plasmid, i.e., a circular double strandedpiece of DNA into which additional DNA segments may be ligated. Forexample, the vector is a viral vector, wherein additional DNA segmentsmay be ligated into the viral genome. In another case, the vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). In another example, thevectors (e.g., non-episomal mammalian vectors) can be integrated intothe genome of a host cell upon introduction into the host cell, andthereby are replicated along with the host genome. Moreover, certainvectors are capable of directing the expression of genes to which theyare operatively linked. Such vectors are referred to herein as“recombinant expression vectors” (or simply, “expression vectors”).

The terms “label” or “labeled” refers to incorporation of anothermolecule in the antibody. For example, the label is a detectable marker,e.g., incorporation of a radiolabeled amino acid or attachment to apolypeptide of biotinyl moieties that can be detected by marked avidin(e.g., streptavidin containing a fluorescent marker or enzymaticactivity that can be detected by optical or colorimetric methods). Inanother embodiment, the label or marker can be therapeutic, e.g., a drugconjugate or toxin. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I) fluorescent labels (e.g., FITC, rhodamine, lanthanidephosphors), enzymatic labels (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescentmarkers, biotinyl groups, predetermined polypeptide epitopes recognizedby a secondary reporter (e.g., leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags),magnetic agents, such as gadolinium chelates, toxins such as pertussistoxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. In some cases, labels are attached by spacer armsof various lengths to reduce potential steric hindrance.

Therapeutic Methods of Use

Also provided are methods for inhibiting IL-6 activity by administeringan IL-6 antibody to a patient in need thereof. Any of the antibodies orantigen-binding portions thereof described herein may be usedtherapeutically. In a preferred embodiment, the IL-6 antibody is ahuman, chimeric or humanized antibody. In another preferred embodiment,the IL-6 is human and the patient is a human patient. Alternatively, thepatient may be a mammal that expresses an IL-6 that the IL-6 antibodycross-reacts with. The antibody may be administered to a non-humanmammal expressing IL-6 purposes or as an animal model of human disease.Such animal models may be used for demonstrating the therapeuticefficacy of the antibodies.

An IL-6 antibody or antibody portion thereof may be administered to apatient who expresses abnormally high levels of IL-6. The antibody maybe administered once, or may be administered multiple times. Theantibody may be administered from three times daily to once every sixmonths or longer. The administering may be on a schedule such as threetimes daily, twice daily, once daily, once every two days, once everythree days, once weekly, once every two weeks, once every month, onceevery two months, once every three months and once every six months. Theantibody may also be administered continuously via a minipump. Theantibody may be administered via a mucosal, buccal, intranasal,inhalable, intravenous, subcutaneous, intramuscular, parenteral, orintratumor route. The antibody may be administered once, at least twiceor for at least the period of time until the condition is treated,palliated or cured. The antibody generally will be administered for aslong as the condition is present. The antibody will generally beadministered as part of a pharmaceutical composition as describedherein. The dosage of antibody will generally be in the range of 0.1 to100 mg/kg, 0.5 to 50 mg/kg, 1 to 20 mg/kg, and 1 to 10 mg/kg. The serumconcentration of the antibody may be measured by any suitable method.

Also provided are methods for the treatment of abnormal cellinfiltration in a mammal, including a human, comprising administering tothe mammal a therapeutically effective amount of an IL-6 antibody orantigen binding portion thereof, as described herein, that is effectivein treating abnormal cell infiltration.

The IL-6 antibodies or antigen-binding portions thereof can be used totreat rheumatoid arthritis. They also can be used to treat otherdiseases in which IL-6 is implicated. Examples of other diseases thatcan be treated using the IL-6 antibodies or antigen binding portionsthereof include osteoarthritis, particularly the pain associated withosteoarthritis, Castleman's disease, juvenile idiopathic arthritis,adult-onset Still's disease, osteoporosis, sepsis, multiple myeloma,renal cell carcinoma, and Crohn's disease.

Some of the diseases that can be treated with the antibodies orantigen-binding portions thereof are discussed below.

Rheumatoid arthritis (RA) is considered a chronic autoimmune andinflammatory disease producing inflamed joints, which eventually swell,become painful, and experience degradation of cartilage, bone, andligaments of the joint. A result of RA is deformity, instability, andstiffness of the joint and scarring within the joint. The jointsdeteriorate at a highly variable rate. Many factors, including geneticpredisposition, may influence the pattern of the disease. People withrheumatoid arthritis may have a mild course, occasional flare-ups withlong periods of remission without disease, or a steadily progressivedisease, which may be slow or rapid. Rheumatoid arthritis may startsuddenly, with many joints becoming inflamed at the same time. Moreoften, it starts subtly, gradually affecting different joints. Usually,the inflammation is symmetric, with joints on both sides of the bodyaffected. Typically, the small joints in the fingers, toes, hands, feet,wrists, elbows, and ankles become inflamed first, followed by the kneesand hips.

Rheumatoid arthritic pain is typically a somatic nociceptive joint pain.Swollen wrists can pinch a nerve and result in numbness or tingling dueto carpal tunnel syndrome. Cysts may develop behind affected knees, canrupture, causing pain and swelling in the lower legs.

Osteoarthritis is characterized by loss of articular cartilage andhypertrophy of bone. Onset of osteoarthritis is usually gradual withpain being a common early symptom. As osteoarthritis progresses, jointmotion diminishes, and tenderness and grating sensations can occur.Osteoarthritis commonly affects the hands, feet, spine, and largeweight-bearing joints, such as the hips and knees. The diagnosis ofosteoarthritis is typically based on symptoms or by X-ray, which canshow narrowing of the joint space, increased density of subchondralbone, formation of osteophytes at the periphery of joints, and formationof pseudocysts in the subchondral marrow. Blood tests are performed toexclude other conditions that can mimic osteoarthritis. In addition, indiagnosing osteoarthritis, arthrocentesis can be performed, whereby asterile needle is used to remove joint fluid. Joint fluid analysis isuseful in excluding gout, infection, and other causes of arthritis.Osteoarthritis is also known as degenerative joint disease, degenerativearthritis, or osteoarthritis.

Reiter's syndrome (reactive arthritis) is inflammation of the joints andtendon attachments at the joints, often accompanied by inflammation ofthe eye's conjunctiva and the mucous membranes, such as those of themouth and genitourinary tract, and by a distinctive rash. Reiter'ssyndrome is also called reactive arthritis because the jointinflammation appears to be a reaction to an infection originating in theintestine or genital tract. This syndrome is most common in men aged 20to 40. There are two forms of Reiter's syndrome: one occurs withsexually transmitted diseases such as a chlamydial infection and theother usually follows an intestinal infection such as shigellosis orsalmonellosis. (Most people who have these infections do not developReiter's syndrome.) People who develop Reiter's syndrome after exposureto these infections appear to have a genetic predisposition to this typeof reaction, related in part to the same gene found in people who haveankylosing spondylitis.

Infectious arthritis is inflammation in a joint resulting frombacterial, fungal, or viral infection of synovial or periarticulartissues. Risk factors for infectious arthritis include advanced age(i.e., greater than 60 years); alcoholism; anemia; arthrocentesis orsurgery; chronic medical illness (e.g., lung or liver disease);diabetes; hemophilia; immunodeficiency, including HIV; immunosuppressivetherapy, including corticosteroids; IV drug use; malignancy; prostheticjoint implant; renal failure; rheumatoid arthritis; sickle cell disease;skin infections; and systemic lupus erythematosus. Patients withrheumatoid arthritis are at particularly increased risk for bacterialarthritis. Joint infections may be acute, with sudden onset of jointpain and swelling (e.g., within a few hours to a few days), or chronic,with insidious development of milder symptoms. Acute bacterial arthritisis commonly accompanied by moderate to severe joint pain, warmth,tenderness, and restricted motion. Chronic bacterial arthritis iscommonly accompanied by gradual swelling, mild warmth, minimal or noredness of the joint area, and aching pain, which may be mild.

Psoriatic arthritis is an inflammatory arthritis affecting the jointsthat occurs in a minority of psoriasis patients and increasingly in someacquired immune deficiency syndrome (AIDS) patients. Psoriatic arthritismay be mild or may produce severe joint deformities resembling jointchanges observed in rheumatoid arthritis. Joints that may be affected bypsoriatic arthritis include distal interphalangeal (DIP) joints offingers and toes, and commonly the asymmetric involvement of large andsmall joints such as sacroiliacs and spine. Psoriasis of the skin ornails may precede or follow joint involvement. The time course ofpsoriatic arthritis is characterized by arthritic exacerbations andremissions that may or may not coincide with skin exacerbations andremissions, and progression to chronic arthritis may occur. Diagnosisincludes a diagnosis of psoriasis, a family history of psoriasis, X-rayfindings showing DIP joint involvement, asymmetric large jointinvolvement, a negative blood test for rheumatoid factor to rule outrheumatoid arthritis, and, in some patients, the presence of HLA-B27antigen, especially when the spine is involved.

Polyarthritis is any type of arthritis which involves five or morejoints. Arthritis of two, three or four joints is called oligoarthritisor pauciarthritis. Polyarthritis is most often caused by an autoimmunedisorder such as rheumatoid arthritis, psoriatic arthritis, or lupuserythematosus, but can also be caused by infections. Polyarthritis maybe experienced at any age and is not gender specific.

Juvenile arthritis is arthritis that begins before age 16. There areseveral different types of juvenile arthritis. The most common type isjuvenile rheumatoid arthritis (JRA), also known as juvenile idiopathicarthritis. JRA includes systemic onset JRA, pauciarticular JRA, whichinvolves fewer than five joints, and polyarticular JRA, which affectsfive or more joints. Diagnosis of JRA involves considering the symptoms,taking x-rays and doing blood analyses. Specific tests that doctor mayuse to diagnose JRA include complete blood counts, blood cultures forinfections, bone marrow examinations, examination of erythrocytesedimentation rate, rheumatoid factor antibody determination,antinuclear antibody determination, and bone scans.

Juvenile rheumatoid arthritis is persistent or recurring inflammation ofthe joints similar to rheumatoid arthritis but beginning before age 16and is characterized by inflammation of joints or connective tissue.There are several types of juvenile rheumatoid arthritis, which aredetermined by the symptoms that develop during the first months of thedisease and how many joints are affected. These types includepauciarticular juvenile rheumatoid arthritis, polyarthritis, andsystemic disease (Still's disease). In pauciarticular juvenilerheumatoid arthritis, four or fewer joints, usually those of the leg areaffected. In polyarthritis, five or more (sometimes as many as 20 to 40)joints are affected. In systemic disease (Still's disease), any numberof joints can be involved.

Juvenile reactive arthritis is persistent or recurring inflammation ofthe joints similar to reactive arthritis but beginning before age 16.

Juvenile psoriatic arthritis is psoriatic arthritis that begins in apatient before the age of 16 years and is characterized by the presenceof chronic arthritis and psoriasis; or chronic arthritis and at leasttwo of the following: dactylitis, nail abnormalities (e.g., pitting oronycholysis), and a family history of psoriasis in at least oneimmediate relative. As in adults with psoriatic arthritis, the arthritismay precede the skin condition. The predominant pattern at onset ofjuvenile psoriatic arthritis is an asymmetric oligoarthritis of smalland large joints often with dactylitis.

In one aspect, the IL-6 mediated disorder is characterized by fibrosis.The term “fibrosis” as used herein refers to a pathological conditioncharacterized by excessive deposition and metabolism of fibroticmaterial (e.g., extracellular matrix) in response to tissue damage. Inmany cases, fibrosis represents a normal repair process (i.e., woundhealing) gone awry due to chronic or excessive tissue insult leading tofibroblast or stellate cell activation and proliferation and collagenaccumulation. Fibrosis conditions include fibroproliferative disordersthat are associated with vascular diseases, such as cardiac disease,cerebral disease, and peripheral vascular disease, as well as all themain tissues and organ systems such as the eye, skin, kidney, lung, gutand liver (Wynn, Nature Reviews 4:583-594 (2004); Bataller, R andBrenner, D., J. Clin. Invest. 115:209-218 (2005)). Other sources arechemotherapeutic drugs, radiation-induced fibrosis, and injuries andburns. While fibrosis conditions cover a wide group of pathologies, itis believed that for most of these conditions, the general mechanismsleading to fibrotic tissue accumulation have many elements in common.Often the condition is initiated in response to an influx ofinflammatory cells and perpetuated by the subsequent cytokine signalingpathways between the infiltrating cells (e.g., macrophages, T cells) andresident cells within the tissue (e.g., stellate, myofibroblast. Kupffercells). In addition, pericytes are a key fibrogenic cell type involvedin the development of scleroderma and PDGF receptor tyrosine kinaseinhibitors (RTKI) have been shown to slow the proliferation of pericytesand suppress skin lesions in patients with this progressive disease. Inthe kidney, leukocyte infiltration plays a major role in mediatingtubulointerstitial inflammation and fibrosis in chronic kidney disease.

As used herein the term “fibrosis” is also used synonymously with“fibroblast accumulation and collagen deposition”. Fibroblasts areconnective tissue cells, which are dispersed in connective tissuethroughout the body. Fibroblasts secrete a nonrigid extracellular matrixcontaining type I and/or type III collagen. In response to an injury toa tissue, nearby fibroblasts or stellate cells migrate into the wound,proliferate, and produce large amounts of collagenous extracellularmatrix. Collagen is a fibrous protein rich in glycine and proline thatis a major component of the extracellular matrix and connective tissue,cartilage, and bone. Collagen molecules are triple-stranded helicalstructures called α-chains, which are wound around each other in aropelike helix. Collagen exists in several forms or types; of these,type I, the most common, is found in skin, tendon, and bone; and typeIII is found in skin, blood vessels, and internal organs. Exemplaryfibrosis conditions include, but are not limited to

-   -   (I) Lung diseases associated with fibrosis, e.g., idiopathic        pulmonary fibrosis, radiation induced fibrosis, chronic        obstructive pulmonary disease (COPD), scleroderma, bleomycin        induced pulmonary fibrosis, chronic asthma, silicosis, asbestos        induced pulmonary fibrosis, acute lung injury and acute        respiratory distress (including bacterial pneumonia induced,        trauma induced, viral pneumonia induced, ventilator induced,        non-pulmonary sepsis induced, and aspiration induced);    -   (II) Chronic nephropathies associated with injury/fibrosis        (kidney fibrosis), e.g., lupus, diabetes, scleroderma,        glomerular nephritis, focal segmental glomerular sclerosis, IgA        nephropathy, hypertension, allograft, Lupus, and Alport;    -   (III) Gut fibrosis, e.g., scleroderma, and radiation induced gut        fibrosis;    -   (IV) Liver fibrosis, e.g., cirrhosis, alcohol induced liver        fibrosis, nonalcoholic steatohepatitis (NASH), biliary duct        injury, primary biliary cirrhosis, infection or viral induced        liver fibrosis (e.g. chronic HCV infection), and autoimmune        hepatitis;    -   (V) Head and neck fibrosis, e.g., radiation induced;    -   (VI) Corneal scarring, e.g., LASIX™, corneal transplant, and        trabeculectomy;    -   (VII) Hypertrophic scarring and keloids, e.g., burn induced and        surgical; and other fibrotic diseases, e.g., sarcoidosis,        scleroderma, spinal cord injury/fibrosis, myelofibrosis,        vascular restenosis, atherosclerosis, Wegener's granulomatosis,        mixed connective tissue disease, and Peyronie's disease.

The term “fibromyalgia” is also known as fibromyalgia syndrome. TheAmerican College of Rheumatology (ACR) 1990 classification criteria forfibromyalgia include a history of chronic, widespread pain for more thanthree months, and the presence of pain at 11 (or more) out of 18 tenderpoints upon physical examination, wherein the tender points occur bothabove and below the waist and on both sides of the body (see e.g., Wolfeet al., Arthritis Rheum., 1990; 33:160-172). Fibromyalgia patientsgenerally display pain perception abnormalities in the form of bothallodynia (pain in response to a normally non-painful stimulus) andhyperalgesia (an increased sensitivity to a painful stimulus). Theeffects of fibromyalgia in a human patient may be assessed using the ACRcriteria, a Fibromyalgia Index Questionnaire (FIQ) total score, indicesof pain severity (e.g., VAS or Likert pain scales) and interference, thenumber of tender points, or a pain threshold assessment.

Although chronic, widespread pain is a hallmark symptom of fibromyalgia,patients typically also exhibit other symptoms, including one or more ofthe following: fatigue, sleep disturbances, migraine or tensionheadaches, irritable bowel syndrome, changes in urinary frequency,morning stiffness, numbness and tingling, dysmenorrhea, multiplechemical sensitivities, difficulty concentrating, and circulatoryproblems that affect the small blood vessels of the skin (Raynaud'sphenomenon). As with many conditions that cause chronic pain,fibromyalgia patients may also experience fibromyalgia-induced anxiety,depression, or both. Some fibromyalgia patients find that cold, dampweather, emotional stress, overexertion, and other factors exacerbatetheir symptoms.

Pain associated with fibromyalgia refers to any pain associated withfibromyalgia syndrome, including the chronic, widespread pain that is ahallmark of fibromyalgia and pain associated with other symptoms offibromyalgia.

Ankylosing spondylitis is a rheumatic disease that causes arthritis ofthe spine and sacroiliac joints. It varies from intermittent episodes ofback pain that occur throughout life to a severe chronic disease thatattacks the spine, peripheral joints and other body organs. Typically,the first symptoms are frequent pain and stiffness in the lower back andbuttocks, which comes on gradually over the course of a few weeks ormonths. The pain is usually dull and diffuse, rather than localized.Ankylosing spondylitis is typically diagnosed with a thorough physicalexam including x-rays, individual medical history, and a family historyof ankylosing spondylitis, as well as blood work including a test forHLA-B27.

Psoriasis is a chronic inflammatory skin disorder that may afflictpeople of all ages. Clinically, psoriasis most frequently affects theskin of the elbows, knees, scalp, lumbosacral areas, intergluteal cleft,or glans penis. Skin affected by psoriasis typically contains one ormore dry lesions comprised of a well-demarcated, pink to salmon-coloredplaque covered by loosely adherent scales that are characteristicallysilver-white in color. In about 30% of psoriasis patients, the nails arealso affected by, for example, pitting or onycholysis. All forms ofpsoriasis are contemplated, including psoriasis annularis and psoriasisannulata, which are also known as psoriasis circinata; psoriasisarthropica; psoriasis diffusa or diffused psoriasis; exfoliativepsoriasis; flexural psoriasis; psoriasis geographica; psoriasis gyrate;psoriasis nummularis; palmar psoriasis; psoriasis punctata; and a rarevariant form known as generalized pustular psoriasis of Zambusch orsimply just pustular psoriasis. Morphologically, established lesions ofpsoriasis have well known histologic characteristics such as epidermalthickening and parakeratotic scale. Pathologically, psoriasis iscurrently believed to be a T-cell mediated autoimmune disorder. Onset ofpsoriasis is usually gradual and the typical time course ischaracterized by chronic remissions and recurrences and, occasionally,acute exacerbations. Diagnosis of psoriasis is made by evaluating apatient's clinical signs and symptoms and family history of psoriasis.Diagnosing psoriasis by just visually inspecting the patient's skinlesions is rarely difficult and usually this is all that is required fora complete diagnosis. Occasionally, however, a skin biopsy is subjectedto a histologic analysis to look for signs of psoriasis.

Systemic lupus erythematosus (“SLE”), also called disseminated lupuserythematosus, is a chronic inflammatory connective tissue disorder ofunknown cause that can involve joints, kidneys, serous surfaces, andvessel walls and that occurs predominantly in young women but also inchildren. Ninety percent of SLE cases occur in women. SLE may beginabruptly with fever, simulating acute infection, or may developinsidiously over months or years with episodes of fever and malaise.Vascular headaches, epilepsy, or psychoses may be initial findings.Manifestations referable to any organ system may appear. Articularsymptoms, ranging from intermittent arthralgias to acute polyarthritis,occur in approximately 90% of patients and may exist for years beforeother manifestations appear. In long-standing disease, capsularinsertional erosions at the metacarpophalangeal joints with markedsecondary joint deformity may occur without x-ray evidence of obviousmarginal erosions (Jaccoud's arthritis). However, most lupuspolyarthritis is nondestructive and nondeforming.

Systemic lupus erythematosus is rare under the age of 5, and mostchildren with SLE develop the disease during adolescence. Signs andsymptoms of juvenile SLE are similar to those in adults. However,children have a particularly high level of transition from the discoidto the systemic disease.

Gout (also known as gouty arthritis) is recurrent acute or chronicarthritis of peripheral joints results from a build-up in the body oftoo much uric acid, which forms crystals that deposit in joints andcause inflammation. During an acute attack of gout there is swelling,inflammation, and extreme pain in a joint, frequently that of the bigtoe. Chronic gout can set in after several years of attacks, permanentlydamaging and deforming joints and destroying cells of the kidney. Mostcases occur in men and the first attack rarely occurs before the age of30.

Undifferentiated Spondyloarthropathy (USpA) is a term used to describesymptoms and signs of spondylitis in someone who does not meet thecriteria for a definitive diagnosis of ankylosing spondylitis or relateddisease. A number of well-established syndromes are included within thespondyloarthropathy family including ankylosing spondylitis, psoriaticarthritis, the arthritis of inflammatory bowel disease, Reiter'ssyndrome, chronic reactive arthritis and enthesitis related juvenilearthritis. Over time, some people with USpA will develop a well-definedform of spondylitis such as ankylosing spondylitis.

Juvenile-onset spondyloarthritis (JSpA), also known as JuvenileSpondyloarthropathy, is the medical term for a group of childhoodrheumatic diseases, which cause arthritis before the age of 16 and mayspan through adult life. The juvenile spondyloarthropathies includeundifferentiated spondyloarthropathy, juvenile ankylosing spondylitis,juvenile psoriatic arthritis, the arthritis associated with inflammatorybowel disease (enteropathogenic arthritis), reactive arthritis,(Reiter's syndrome is one type of reactive arthritis), and the SEAsyndrome (seronegativity, enthesopathy, arthropathy). JSpA typicallycauses pain and inflammation in the joints in the lower part of thebody, for example, the pelvis, hips, knees and ankles. Other areas ofthe body can also be affected, such as the spine, eyes, skin and bowels.Fatigue and lethargy can also occur.

Crohn's disease is a nonspecific chronic transmural inflammatory diseasethat most commonly affects the distal ileum and colon but may occur inany part of the GI tract. Chronic diarrhea with abdominal pain, fever,anorexia, weight loss, and a right lower quadrant mass or fullness arethe most common symptoms of Crohn's disease. Less common symptomsinclude poor appetite, fever, night sweats, rectal pain, and rectalbleeding. Crohn's disease may affect the colon, the rectum, and thesmall intestine and, in rare instances, also the stomach, mouth, andesophagus. The most common patterns of Crohn's disease pathology are (1)inflammation characterized by right lower quadrant abdominal pain andtenderness; (2) recurrent partial obstruction caused by intestinalstenosis and leading to severe colic, abdominal distention,constipation, and vomiting; (3) diffuse jejunoileitis, with inflammationand obstruction resulting in malnutrition and chronic debility; and (4)abdominal fistulas and abscesses, usually late developments, oftencausing fever, painful abdominal masses, and generalized wasting.Crohn's disease should be suspected in a patient with the inflammatoryor obstructive symptoms described above and in a patient withoutprominent GI symptoms but with perianal fistulas or abscesses or withotherwise unexplained arthritis, erythema nodosum, fever, anemia, orstunted growth (in a child). Laboratory findings are nonspecific and mayinclude anemia, leukocytosis, hypoalbuminemia, and increased levels ofacute-phase reactants reflected in elevated ESR, C-reactive protein, ororosomucoids. Elevated alkaline phosphatase and γ-glutamyltranspeptidase accompanying colonic disease often reflect primarysclerosing cholangitis. Diagnosis is usually made by x-ray.

In advanced cases, the string sign may be seen with marked ilealstrictures and separation of bowel loops. In earlier cases, x-raydiagnosis may sometimes be difficult, but air double-contrast bariumenema and enteroclysis may show superficial aphthous and linear ulcers.Colonoscopy and biopsy may help confirm the diagnosis of Crohn's colitisand allow direct visualization and biopsy of the terminal ileum. UpperGI endoscopy may identify gastroduodenal involvement in Crohn's diseasepatients with upper GI symptoms.

Ulcerative colitis is a chronic, inflammatory, and ulcerative diseasearising in the colonic mucosa, characterized most often by bloodydiarrhea. Bloody diarrhea of varied intensity and duration isinterspersed with asymptomatic intervals are the most common symptoms ofulcerative colitis. Usually an attack begins insidiously, with increasedurgency to defecate, mild lower abdominal cramps, and blood and mucus inthe stools. However, an attack may be acute and fulminant, with suddenviolent diarrhea, high fever, signs of peritonitis, and profoundtoxemia. Some cases develop following a documented infection (e.g.,amebiasis, bacillary dysentery). When ulceration is confined to therectosigmoid, the stool may be normal or hard and dry, but rectaldischarges of mucus loaded with red blood cells and white blood cellsaccompany or occur between bowel movements. Systemic symptoms are mildor absent. If ulceration extends proximally, stools become looser andthe patient may have >10 bowel movements/day, often with severe crampsand distressing rectal tenesmus, without respite at night. The stoolsmay be watery, may contain mucus, and frequently consist almost entirelyof blood and pus. Malaise, fever, anemia, anorexia, weight loss,leukocytosis, and hypoalbuminemia may be present with extensive activeulcerative colitis. The patient's history and stool examination permit apresumptive diagnosis of ulcerative colitis that should always beconfirmed by sigmoidoscopy, which provides a direct, immediateindication of disease activity. In early cases, the mucous membrane isfinely granular and friable, with loss of the normal vascular patternand often with scattered hemorrhagic areas; minimal trauma (friability)causes bleeding in multiple pinpoint spots. The mucosa soon breaks downinto a red, spongy surface dotted with many tiny blood- and pus-oozingulcers. As the mucosa becomes progressively involved, the inflammationand hemorrhage extend into the bowel muscle. Large mucosal ulcers withcopious purulent exudate characterize severe disease. Islands ofrelatively normal or hyperplastic inflammatory mucosa (pseudopolyps)project above areas of ulcerated mucosa. Biopsies may be nonspecific andsometimes cannot exclude acute infectious (self-limited) colitis;however, features that suggest chronicity (e.g., distorted cryptarchitecture, crypt atrophy, a chronic inflammatory infiltrate) supportthe diagnosis of ulcerative colitis. Even during asymptomatic intervals,the sigmoidoscopic appearance is rarely normal; some degree offriability or granularity almost always persists. There is loss of thenormal vascular pattern, and biopsy shows evidence of chronicinflammation. Plain x-rays of the abdomen sometimes help to judge theseverity and proximal extent of the colitis by showing loss ofhaustration, mucosal edema, and absence of formed stool in the diseasedbowel. Later in the course of disease, however, the entire colon shouldbe evaluated to determine the extent of involvement. Total colonoscopyis the most sensitive and widely used method, although barium enema canbe informative. Colonoscopy with biopsy is mandatory to evaluate thenature of a stricture. Biopsy may also help distinguish ulcerativecolitis from Crohn's disease if the inflammation is highly focal or if agranuloma is seen.

Irritable bowel syndrome (IBS) is a motility disorder involving theentire GI tract, causing recurring upper and lower GI symptoms,including variable degrees of abdominal pain, constipation and/ordiarrhea, and abdominal bloating. The cause of irritable bowel syndrome(IBS) is unknown. No anatomic cause can be found. Emotional factors,diet, drugs, or hormones may precipitate or aggravate heightened GImotility. Features of IBS are pain relieved by defecation, analternating pattern of bowel habits, abdominal distention, mucus in thestool, and sensation of incomplete evacuation after defecation. Ingeneral, the character and location of pain, precipitating factors, anddefecatory pattern are distinct for each patient. Patients with IBS mayalso have extraintestinal symptoms (e.g., fibromyalgia, headaches,dyspareunia, temporomandibular joint syndrome). Two major clinical typesof IBS have been described. In constipation-predominant IBS,constipation is common, but bowel habits vary. Most patients have painover at least one area of the colon, associated with periodicconstipation alternating with a more normal stool frequency. Stool oftencontains clear or white mucus. The pain is either colicky, coming inbouts, or a continuous dull ache; it may be relieved by a bowelmovement. Eating commonly triggers symptoms. Bloating, flatulence,nausea, dyspepsia, and pyrosis can also occur. Diarrhea-predominant IBSis characterized by precipitous diarrhea that occurs immediately onrising or during or immediately after eating. Nocturnal diarrhea isunusual. Pain, bloating, and rectal urgency are common, and incontinencemay occur. Diagnosis of IBS is based on characteristic bowel patterns,time and character of pain, and exclusion of other disease processesthrough physical examination and routine diagnostic tests. Due to a lackof a readily identifiable structural or biochemical abnormality in thissyndrome, the medical community has developed a consensus definition andcriteria, known as the Rome criteria, to aid in diagnosis of IBS.According to the Rome criteria, IBS is indicated by abdominal pain ordiscomfort which is (1) relieved by defection and/or (2) associated witha change in frequency or consistency of stools, plus two or more of thefollowing: altered stool frequency, altered stool formation, alteredstool passage, passage of mucus, and bloating or feeling of abdominaldistention. Palpation of the abdomen may reveal tenderness, particularlyin the left lower quadrant, at times associated with a palpable, tendersigmoid. A routine digital rectal examination should be performed on allpatients, and a pelvic examination on women.

Irritable bowel disease (IBD), also known as inflammatory bowel disease,is characterized by chronic inflammation at various sites in the GItract. IBD comprises two known clinical subtypes, Crohn's Disease (CD)and ulcerative colitis (UC). Certain differences in disease patternsjustify a distinction between Crohn's disease and ulcerative colitis.

Pain associated with IBD and IBS may present as either chronic or acutepain. For example a feature of IBS is acute pain relieved by defecationwhile chronic abdominal pain is typical of Crohn's disease. While painassociated with IBD and IBS may occur extraintestinal or extravisceralgenerally these ailments produce visceral pain. Visceral pain is painassociated with the viscera, which encompasses the organs of theabdominal cavity. These organs include the sex organs, spleenintestines, colon, rectum and other organs of the digestive system.Visceral pain has five important clinical characteristics: (1) it is notevoked from all viscera (organs such as liver, kidney, most solidviscera, and lung parenchyma are not sensitive to pain); (2) it is notalways linked to visceral injury; (3) it is diffuse and poorlylocalized; (4) it is referred to other locations; and (5) it isaccompanied with motor and autonomic reflexes, such as the nausea,vomiting, and lower-back muscle tension that occurs in renal colic.

Pain is an important physiological protective mechanism designed to warnof danger from potentially injurious stimuli from the externalenvironment. The system operates through a specific set of primarysensory neurones and is activated by noxious stimuli via peripheraltransducing mechanisms (see Millan, 1999, Prog. Neurobiol., 57, 1-164for a review). These sensory fibers are known as nociceptors and arecharacteristically small diameter axons with slow conduction velocities.Nociceptors encode the intensity, duration and quality of noxiousstimulus and by virtue of their topographically organized projection tothe spinal cord, the location of the stimulus. The nociceptors are foundon nociceptive nerve fibers of which there are two main types, A-deltafibers (myelinated) and C fibers (non-myelinated). The activitygenerated by nociceptor input is transferred, after complex processingin the dorsal horn, either directly, or via brain stem relay nuclei, tothe ventrobasal thalamus and then on to the cortex, where the sensationof pain is generated.

Pain may generally be classified as acute or chronic. Acute pain beginssuddenly and is short-lived (usually twelve weeks or less). It isusually associated with a specific cause such as a specific injury andis often sharp and severe. It is the kind of pain that can occur afterspecific injuries resulting from surgery, dental work, a strain or asprain. Acute pain does not generally result in any persistentpsychological response. In contrast, chronic pain is long-term pain,typically persisting for more than three months and leading tosignificant psychological and emotional problems. Common examples ofchronic pain are neuropathic pain (e.g. painful diabetic neuropathy,postherpetic neuralgia), carpal tunnel syndrome, back pain, headache,cancer pain, arthritic pain and chronic post-surgical pain.

When a substantial injury occurs to body tissue, via disease or trauma,the characteristics of nociceptor activation are altered and there issensitisation in the periphery, locally around the injury and centrallywhere the nociceptors terminate. These effects lead to a heightenedsensation of pain. In acute pain these mechanisms can be useful, inpromoting protective behaviors which may better enable repair processesto take place. The normal expectation would be that sensitivity returnsto normal once the injury has healed. However, in many chronic painstates, the hypersensitivity far outlasts the healing process and isoften due to nervous system injury. This injury often leads toabnormalities in sensory nerve fibers associated with maladaptation andaberrant activity (Woolf & Salter, 2000, Science, 288, 1765-1768).

Clinically, pain is present when discomfort and abnormal sensitivityfeature among the patient's symptoms. Patients tend to be quiteheterogeneous and may present with various pain symptoms. Such symptomsinclude: 1) spontaneous pain which may be dull, burning, or stabbing; 2)exaggerated pain responses to noxious stimuli (hyperalgesia); and 3)pain produced by normally innocuous stimuli (allodynia—Meyer et al.,1994, Textbook of Pain, 13-44). Although patients suffering from variousforms of acute and chronic pain may have similar symptoms, theunderlying mechanisms may be different and may, therefore, requiredifferent treatment strategies. Pain can also therefore be divided intoa number of different subtypes according to differing pathophysiology,including nociceptive, inflammatory and neuropathic pain.

Nociceptive pain is induced by tissue injury or by intense stimuli withthe potential to cause injury. Pain afferents are activated bytransduction of stimuli by nociceptors at the site of injury andactivate neurons in the spinal cord at the level of their termination.This is then relayed up the spinal tracts to the brain where pain isperceived (Meyer et al., 1994, Textbook of Pain, 13-44). The activationof nociceptors activates two types of afferent nerve fibers. MyelinatedA-delta fibers transmit rapidly and are responsible for sharp andstabbing pain sensations, while unmyelinated C fibers transmit at aslower rate and convey a dull or aching pain. Moderate to severe acutenociceptive pain is a prominent feature of pain from central nervoussystem trauma, strains/sprains, burns, myocardial infarction and acutepancreatitis, post-operative pain (pain following any type of surgicalprocedure), posttraumatic pain, renal colic, cancer pain and back pain.Cancer pain may be chronic pain such as tumor related pain (e.g. bonepain, headache, facial pain or visceral pain) or pain associated withcancer therapy (e.g. postchemotherapy syndrome, chronic postsurgicalpain syndrome or post radiation syndrome). Cancer pain may also occur inresponse to chemotherapy, immunotherapy, hormonal therapy orradiotherapy. Back pain may be due to herniated or rupturedintravertebral discs or abnormalities of the lumber facet joints,sacroiliac joints, paraspinal muscles or the posterior longitudinalligament. Back pain may resolve naturally but in some patients, where itlasts over 12 weeks, it becomes a chronic condition which can beparticularly debilitating.

Neuropathic pain is currently defined as pain initiated or caused by aprimary lesion or dysfunction in the nervous system. Nerve damage can becaused by trauma and disease and thus the term ‘neuropathic pain’encompasses many disorders with diverse aetiologies. These include, butare not limited to, peripheral neuropathy, diabetic neuropathy, postherpetic neuralgia, trigeminal neuralgia, back pain, cancer neuropathy,HIV neuropathy, phantom limb pain, carpal tunnel syndrome, centralpost-stroke pain and pain associated with chronic alcoholism,hypothyroidism, uremia, multiple sclerosis, spinal cord injury,Parkinson's disease, epilepsy and vitamin deficiency. Neuropathic painis pathological as it has no protective role. It is often present wellafter the original cause has dissipated, commonly lasting for years,significantly decreasing a patient's quality of life (Woolf and Mannion,1999, Lancet, 353, 1959-1964). The symptoms of neuropathic pain aredifficult to treat, as they are often heterogeneous even betweenpatients with the same disease (Woolf & Decosterd, 1999, Pain Supp., 6,S141-S147; Woolf and Mannion, 1999, Lancet, 353, 1959-1964). Theyinclude spontaneous pain, which can be continuous, and paroxysmal orabnormal evoked pain, such as hyperalgesia (increased sensitivity to anoxious stimulus) and allodynia (sensitivity to a normally innocuousstimulus).

The inflammatory process is a complex series of biochemical and cellularevents, activated in response to tissue injury or the presence offoreign substances, which results in swelling and pain (Levine andTaiwo, 1994, Textbook of Pain, 45-56). Arthritic pain is the most commoninflammatory pain. Rheumatoid disease is one of the commonest chronicinflammatory conditions in developed countries and rheumatoid arthritisis a common cause of disability. The exact aetiology of rheumatoidarthritis is unknown, but current hypotheses suggest that both geneticand microbiological factors may be important (Grennan & Jayson, 1994,Textbook of Pain, 397-407). It has been estimated that almost 16 millionAmericans have symptomatic osteoarthritis (OA) or degenerative jointdisease, most of whom are over 60 years of age, and this is expected toincrease to 40 million as the age of the population increases, makingthis a public health problem of enormous magnitude (Houge & Mersfelder,2002, Ann Pharmacother., 36, 679-686; McCarthy et al., 1994, Textbook ofPain, 387-395). Most patients with osteoarthritis seek medical attentionbecause of the associated pain. Arthritis has a significant impact onpsychosocial and physical function and is known to be the leading causeof disability in later life. Ankylosing spondylitis is also a rheumaticdisease that causes arthritis of the spine and sacroiliac joints. Itvaries from intermittent episodes of back pain that occur throughoutlife to a severe chronic disease that attacks the spine, peripheraljoints and other body organs.

Another type of inflammatory pain is visceral pain which includes painassociated with inflammatory bowel disease (IBD). Visceral pain is painassociated with the viscera, which encompass the organs of the abdominalcavity. These organs include the sex organs, spleen and part of thedigestive system. Pain associated with the viscera can be divided intodigestive visceral pain and non-digestive visceral pain. Commonlyencountered gastrointestinal (GI) disorders that cause pain includefunctional bowel disorder (FBD) and inflammatory bowel disease (IBD).These GI disorders include a wide range of disease states that arecurrently only moderately controlled, including, in respect of FBD,gastro-esophageal reflux, dyspepsia, irritable bowel syndrome (IBS) andfunctional abdominal pain syndrome (FAPS), and, in respect of IBD,Crohn's disease, ileitis and ulcerative colitis, all of which regularlyproduce visceral pain. Other types of visceral pain include the painassociated with dysmenorrhea, cystitis and pancreatitis and pelvic pain.

It should be noted that some types of pain have multiple aetiologies andthus can be classified in more than one area, e.g. back pain and cancerpain have both nociceptive and neuropathic components.

Other types of pain include:

-   -   pain resulting from musculo-skeletal disorders, including        myalgia, fibromyalgia, spondylitis, sero-negative        (non-rheumatoid) arthropathies, non-articular rheumatism,        dystrophinopathy, glycogenolysis, polymyositis and pyomyositis;    -   heart and vascular pain, including pain caused by angina,        myocardical infarction, mitral stenosis, pericarditis, Raynaud's        phenomenon, scleredoma and skeletal muscle ischemia;    -   head pain, such as migraine (including migraine with aura and        migraine without aura), cluster headache, tension-type headache        mixed headache and headache associated with vascular disorders;        and    -   orofacial pain, including dental pain, otic pain, burning mouth        syndrome and temporomandibular myofascial pain.

IL-6 antibodies or antigen-binding portions thereof can be used incombination with one or more other therapeutic agents. For example, anantibody or antigen-binding portions thereof can be used with a COX-2inhibitor, such as celecoxib, for the treatment of diseases such asrheumatoid arthritis, osteoarthritis and pain. IL-6 antibodies orantigen-binding portions thereof and the other therapeutic agents can beadministered to the patient in the same dosage form or in differentdosage forms. Moreover, they can be administered at the same time or atdifferent times. Below are some examples of therapeutic agents that canbe used in combination with anti-IL-6 antibodies or antigen-bindingportions thereof.

Rheumatoid Arthritis

IL-6 antibodies and antigen binding portions thereof may also be used inco-therapies. Suitable antiinflammatory co-therapy compounds include:cyclosporine, zoledronic acid, efalizumab, alefacept, etodolac,lornoxicam, OM-89, valdecoxib, tocilizumab, abatacept, meloxicam,etanercept, nambumetone, rimexolone, 153Sm-EDTMP, prosorba, imidazolesalicylate, oprelvekin, hylauronic acid, naproxen, piroxicam, diacerein,lumericoxib, tacrolimus, aceclofenac, actarit, tenoxicam, rosiglitazone,deflazacort, adalimumab, leflunomide, risedronate sodium, misoprostoland diclofenac, SK-1306X, infliximab, anakinra, celecoxib, diclofenac,etoricoxib and felbinac, reumacon, golimumab, denosumab, ofatumumab,10rT1 antibody, pelubiprofen, licofelone, temsirolimus, eculizumab,iguratimod, and prednisone. Other suitable antiinflammatories includeCP-481715, ABN-912, MLN-3897, HuMax-IL-15, RA-1, paclitaxel, Org-37663,Org 39141, AED-9056, AMG-108, fontolizumab, pegsunercept, pralnacasan,apilimod, GW-274150, AT-001, 681323 (GSK) K-832, R-1503, ocrelizumab,DE-096, Cpn10, THC+CBD (GW Pharma), 856553 (GSK), ReN-1869,immunoglobulin, mm-093, amelubant, SCIO-469, ABT-874, LenkoVAX,LY-2127399, TRU-015, KC-706, amoxapinet and dipyridamole, TAK-715, PG760564, VX-702, prednisolone and dipyridamole, PMX-53, belimumab,prinaberel, CF-101, tgAAV-TNFR:Fc, R-788, prednisolone and SSRI,CP-690550 and PMI-001.

Osteoarthritis

IL-6 antibodies and antigen binding portions thereof may further beco-administered for the treatment of osteoarthritis with one or moreagents useful for treating one or more indicia of osteoarthritis.Examples of agents useful for treating one or more indicia ofosteoarthritis to be used in combination with anti-IL-6 antibodies orantigen-binding portions thereof include matrix metalloproteinase (MMP)inhibitors, aggrecanase inhibitors, inducible nitric oxide (iNOS)inhibitors, inhibitors of insulin-like growth factor (IGF) expression oractivity, inhibitors of fibroblast growth factor (FGF) expression oractivity, inhibitors of CD 44 expression or activity, inhibitors ofinterleukin (IL) expression or activity, inhibitors of tumor necrosisfactor alpha (TNF-alpha) expression or activity, inhibitors of tumornecrosis factor-inducible protein 6 (TSG-6) expression or activity,inhibitors of Bikunin expression or activity, inhibitors ofbeta-secretase (BACE), inhibitors of PACE-4, inhibitors of nuclearreceptor rev-ErbA alpha (NR1D1) expression or activity, inhibitors ofendothelial differentiation sphingolipid G-protein-coupled receptor 1(EDG-1) expression or activity, inhibitors of proteinase-activatedreceptor (PAR) expression or activity, inhibitors of cartilage-derivedretinoic-acid-sensitive protein (CD-RAP) expression or activity,inhibitors of protein kinase C zeta (PKCz), inhibitors of resistinexpression or activity, inhibitors of a disintegrin andmetalloproteinase 8 (ADAMS), inhibitors of complement component 1 ssubcomponent (C1s) expression or activity, inhibitors of formyl peptidereceptor-like 1 (FPRL1) expression or activity.

Additional examples of agents useful in combination with IL-6 antibodiesand antigen binding portions thereof include inhibitors of MMP-2, -3,-9, or -13; inhibitors of aggrecanase-1 or -2; inhibitors of IGF-1 or -2expression or activity; inhibitors of FGF-2, -18, or -9 expression oractivity; and inhibitors of IL-1, -4 or -6 expression or activity.

Further examples of agents useful in combination with IL-6 antibodiesand antigen binding portions thereof include IGF-1 or -2 antibodies; FGFreceptor-2 or -3 antagonists, CD 44 antibodies, IL-1, -4 or -6antibodies, TNF-alpha antibodies; TSG-6 antibodies; bikunin antibodies;NR1D1 antagonists; EDG-1 antagonists; PAR antagonists, CD-RAPantibodies, resistin antibodies, C1s antibodies, and FPRL1 antibodies.

Pain

IL-6 antibodies or antigen binding portions thereof can be administeredin combination with one or more additional pharmacologically activecompounds for the treatment of pain. The compounds may be administeredat the same time in a single dosage form or separately in dosage formsthat can be the same or different. Alternatively, the compounds can beadministered sequentially. The pharmaceutically acceptable salts of thepharmacologically active compounds may also be used in the combinations.

Examples of compounds that can be administered with IL-6 antibodies orantigen binding portions thereof include:

Cyclooxygenase-2 (COX-2) selective inhibitors such as celecoxib,rofecoxib, parecoxib, valdecoxib, deracoxib, etoricoxib, andlumiracoxib; opioid analgesics such as morphine, hydromorphone,oxymorphone, fentanyl, codeine, dihydrocodeine, oxycodone, hydrocodone,buprenorphine, tramadol, and nalbuphine; nonsteroidal antiinflammatorydrugs (NSAIDs) such as aspirin, diclofenac, diflunisal, ibuprofen,fenoprofen, naproxen, nepafenac, and acetaminophen; Phosphodiesterase Vinhibitors (PDEV) such as sildenafil; alpha-2-delta ligands such asgabapentin and pregabalin; and local anaesthetics such as benzocaine,lidocaine, ropivacaine, menthol, camphor and methyl salicylate.

Examples of other types of compounds and classes of compounds that canbe used in combination with IL-6 antibodies and antigen binding portionsthereof include: barbiturate sedatives; benzodiazepines; Histamine H₁antagonists having a sedative action; sedatives; skeletal musclerelaxants; N-methyl-D-aspartic acid (NMDA) receptor antagonists;alpha-adrenergics; tricyclic antidepressants; anticonvulsants such ascarbamazepine; tachykinin (NK) antagonists, particularly NK-3, NK-2 orNK-1 antagonists; muscarinic antagonists; neuroleptics; vanilloidreceptor agonists or antagonists; beta-adrenergics; corticosteroids;Serotonin (5-HT) receptor agonists or antagonists such as a5-HT_(1B/1D), 5-HT_(2A), and 5-HT₃ receptor antagonists; cholinergic(nicotinic) analgesics; cannabinoids; metabotropic glutamate subtype 1receptor (mGluR1) antagonists; serotonin reuptake inhibitors such assertraline; noradrenaline (norepinephrine) reuptake inhibitors such asreboxetine, in particular (S,S)-reboxetine; dual serotonin-noradrenalinereuptake inhibitors such as duloxetine; inducible nitric oxide synthase(iNOS) inhibitors such asS-[2-[(1-iminoethyl)amino]ethyl]L-homocysteine,S-[2-[(1-iminoethyl)-amino]ethyl]-4,4-dioxo-L-cysteine,S-[2-[(1-iminoethyl)amino]ethyl]-2-methyl-L-cysteine,(2S,5Z)-2-amino-2-methyl-7-[(1-iminoethyl)amino]-5-heptenoic acid,2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)-butyl]thio]-5-chloro-3-pyridinecarbonitrile;2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-4-chlorobenzonitrile,(2S,4R)-2-amino-4-[[2-chloro-5-(trifluoromethyl)phenyl]thio]-5-thiazolebutanol,2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-6-(trifluoromethyl)-3pyridinecarbonitrile,2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-5-chlorobenzonitrile,N-[4-[2-(3-chlorobenzylamino)ethyl]phenyl]thiophene-2-carboxamidine, andguanidinoethyldisulfide; acetylcholinesterase inhibitors; prostaglandinE₂ subtype 4 (EP4) antagonists such asN-[({2-[4-(2-ethyl-4,6-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl)phenyl]ethyl}amino)-carbonyl]-4-methylbenzenesulfonamideor4-[(1S)-1-({[5-chloro-2-(3-fluorophenoxyl)pyridin-3-yl]carbonyl}amino)ethyl]benzoicacid; leukotriene B4 antagonists such as1-(3-biphenyl-4-ylmethyl-4-hydroxy-chroman-7-yl)-cyclopentanecarboxylicacid; 5-lipoxygenase inhibitors; and sodium channel blockers.

Fibromyalgia Syndrome

Products: analgesics such as acetaminophen, naproxen sodium, ibuprofen,tramadol, trazodone; cyclobenzaprine; aspirin, celecoxib, valdecoxib,indomethacin, and other NSAIDs; antidepressants such as tricyclicantidepressants and selective serotonin reuptake inhibitors, for exampleantidepressants such as amitriptyline, imipramine, nortriptyline,doxepin, fluoxetine, sertraline, and paroxetine; muscle relaxants suchas cyclobenzaprine; sleeping aids such as zolpidem.

Classes: norepinephrine-serotonin reuptake inhibitors (NSRIs and SNRIs);norepinephrine reuptake inhibitor (NRIs); selective serotonin reuptakeinhibitors (SSRIs); tricyclic antidepressants; selectivecyclooxygenase-2 (COX-2) inhibitors; nonsteroidal anti-inflammatorydrugs (NSAIDs); analgesics.

Ankylosing Spondylitis

Products: analgesics such as acetaminophen, naproxen sodium, ibuprofen,tramadol, aspirin, celecoxib, valdecoxib, indomethacin, and otherNSAIDs; disease-modifying antirheumatic drugs (DMARDs) such assulfasalazine or methotrexate; corticosteroids; and tumor necrosisfactor (TNF) blockers such as etanercept and infliximab.

Classes: analgesics; NSAIDs; COX-2 inhibitors; DMARDs; corticosteroids;TNF blockers.

Psoriasis

Products: phototherapy, including psoralen ultraviolet A (psoralen UVAor PUVA) therapy, narrow-band ultraviolet B (UVB) therapy, andcombination light therapy; topical corticosteroids; vitamin D analogssuch as calcipotriene; anthralin; topical retinoids (i.e., vitamin Aderivatives) such as acitretin and tazarotene; clobetasol propionate;methotrexate; azathioprine; cyclosporine; hydroxyurea; andimmune-modulating drugs such as alefacept, efalizumab, and etanercept.

Classes: phototherapy; corticosteroids; vitamin D analogs; vitamin Aderivatives.

Gout

Products: NSAIDs such as acetaminophen, naproxen sodium, ibuprofen,tramadol, aspirin, celecoxib, valdecoxib, and indomethacin; andcorticosteroids such as prednisone.

Classes: analgesics; NSAIDs; COX-2 inhibitors; and corticosteroids.

Crohn's Disease

Products: analgesics such as acetaminophen, naproxen sodium, ibuprofen,tramadol, aspirin, celecoxib, valdecoxib, indomethacin, and otherNSAIDs; anti-inflammatory drugs; sulfasalazine, mesalamine, balsalazide,and olsalazine; and corticosteroids such as prednisone and budesonide;immunosuppressant drugs such as azathioprine, mercaptopurine, TNFblockers such as infliximab and adalimumab, methotrexate, andcyclosporine; antibiotics such as metronidazole and ciprofloxacin;anti-diarrheals such as loperamide; and laxatives.

Classes: analgesics; NSAIDs; COX-2 inhibitors; anti-inflammatory drugs;TNF blockers; antibiotics; anti-diarrheals; and laxatives.

Ulcerative Colitis

Classes: analgesics such as acetaminophen, naproxen sodium, ibuprofen,tramadol, aspirin, celecoxib, valdecoxib, indomethacin, and otherNSAIDs; anti-inflammatory drugs; sulfasalazine, mesalamine, balsalazide,and olsalazine; corticosteroids; immunosuppressant drugs such asazathioprine, mercaptopurine, TNF blockers such as infliximab andadalimumab, methotrexate, and cyclosporine; anti-diarrheals such asloperamide; and laxatives.

Classes: NSAIDs; COX-2 inhibitors; anti-inflammatory drugs; TNFblockers; corticosteroids; immunosuppressants; Janus kinase-3 (Jak-3)inhibitors; TNF blockers; anti-diarrheals; and laxatives.

Irritable Bowel Syndrome

Products: anti-diarrheals such as loperamide; laxatives; anticholinergicdrugs; antidepressants such as tricyclic antidepressants and selectiveserotonin reuptake inhibitors, for example antidepressants such asamitriptyline, imipramine, nortriptyline, doxepin, fluoxetine,sertraline, and paroxetine; alosetron; and tegaserod.

Classes: anti-diarrheals; laxatives; anticholinergic drugs;norepinephrine-serotonin reuptake inhibitors (NSRIs and SNRIs);norepinephrine reuptake inhibitor (NRIs); selective serotonin reuptakeinhibitors (SSRIs); tricyclic antidepressants.

Pharmaceutical Compositions and Administration

Also provided are pharmaceutical compositions for the treatment ofabnormal cell infiltration in a mammal, including a human, comprising anamount of an IL-6 antibody or antigen binding portion thereof, asdescribed herein, that is effective in treating abnormal cellinfiltration, and a pharmaceutically acceptable carrier. Thecompositions provide a therapeutic benefit to patients with one of moreof a variety of inflammatory and autoimmune diseases, such as rheumatoidarthritis, atherosclerosis, granulomatous diseases, multiple sclerosis,asthma and cancer.

IL-6 antibodies and antigen-binding portions thereof can be incorporatedinto pharmaceutical compositions suitable for administration to asubject. Typically, the pharmaceutical composition comprises an IL-6antibody or antigen-binding portion thereof and a pharmaceuticallyacceptable carrier. As used herein, “pharmaceutically acceptablecarrier” means any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. Some examplesof pharmaceutically acceptable carriers are water, saline, phosphatebuffered saline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Additional examples ofpharmaceutically acceptable substances are wetting agents or minoramounts of auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the antibody.

The compositions of this invention may be in a variety of forms, forexample, liquid, semi-solid and solid dosage forms, such as liquidsolutions (e.g., injectable and infusible solutions), dispersions orsuspensions, tablets, pills, powders, liposomes and suppositories. Theform depends on the intended mode of administration and therapeuticapplication. Typical compositions are in the form of injectable orinfusible solutions, such as compositions similar to those used forpassive immunization of humans. In one case the mode of administrationis parenteral (e.g., intravenous, subcutaneous, intraperitoneal,intramuscular). In another case, the antibody is administered byintravenous infusion or injection. In another case, the antibody isadministered by intramuscular or subcutaneous injection. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with or without an added preservative. Thecompositions may take such forms as suspensions, solutions, or emulsionsin oily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use. Sterileinjectable solutions can be prepared by incorporating the IL-6 antibodyin the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfilter sterilization. In one case, the antibody is administered in aformulation as a sterile aqueous solution having a pH that ranges fromabout 5.0 to about 6.5 and comprising from about 1 mg/ml to about 200mg/ml of antibody, from about 1 millimolar to about 100 millimolar ofhistidine buffer, from about 0.01 mg/ml to about 10 mg/ml of polysorbate80 or polysorbate 20, from about 100 millimolar to about 400 millimolarof a non-reducing sugar selected from but not limited to trehalose orsucrose, from about 0.01 millimolar to about 1.0 millimolar of disodiumEDTA dihydrate and optionally comprise a pharmaceutically acceptableantioxidant in addition to a chelating agent. Suitable antioxidantsinclude, but are not limited to, methionine, sodium thiosulfate,catalase, and platinum. For example, the composition may containmethionine in a concentration that ranges from 1 mM to about 100 mM, andin particular, is about 27 mM. In some cases, a formulation contains 5mg/ml of antibody in a buffer of 20 mM sodium citrate, pH 5.5, 140 mMNaCl, and 0.2 mg/ml polysorbate 80. It is to be noted that dosage valuesmay vary with the type and severity of the condition to be alleviated.In the case of sterile powders for the preparation of sterile injectablesolutions, the suitable methods of preparation include vacuum drying andfreeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

IL-6 antibodies or antigen-binding portions thereof can be administeredby a variety of methods, although for many therapeutic applications, theroute/mode of administration can be subcutaneous, intramuscular, orintravenous infusion. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults.

In certain cases, the IL-6 antibody compositions may be prepared with acarrier that will protect the antibody against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations may be used. See, e.g.,Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson,ed., Marcel Dekker, Inc., New York, 1978, which is incorporated hereinby reference.

Additional active compounds also can be incorporated into thecompositions. In some cases, an inhibitory IL-6 antibody isco-formulated with and/or co-administered with one or more additionaltherapeutic agents. These agents include, without limitation, antibodiesthat bind other targets, anti-tumor agents, anti-angiogenesis agents,signal transduction inhibitors, anti-proliferative agents,chemotherapeutic agents, or peptide analogues that inhibit IL-6. Suchcombination therapies may require lower dosages of the inhibitory IL-6antibody as well as the co-administered agents, thus avoiding possibletoxicities or complications associated with the various monotherapies.

The compositions may include a “therapeutically effective amount” or a“prophylactically effective amount” of an antibody or antigen-bindingportion. A “therapeutically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired therapeutic result. A therapeutically effective amount of theantibody or antigen-binding portion may vary according to factors suchas the disease state, age, sex, and weight of the individual, and theability of the antibody or antibody portion to elicit a desired responsein the individual. A therapeutically effective amount is also one inwhich any toxic or detrimental effects of the antibody orantigen-binding portion are outweighed by the therapeutically beneficialeffects. A “prophylactically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired prophylactic result. Typically, since a prophylactic dose isused in subjects prior to or at an earlier stage of disease, theprophylactically effective amount may be less than the therapeuticallyeffective amount.

Dosage regimens can be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus can be administered, several divided doses can be administeredover time or the dose can be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of IL-6 antibody or antigen bindingportion thereof calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an IL-6 antibody or antibodyportion is 0.025 to 50 mg/kg, 0.1 to 50 mg/kg, 0.1-25, 0.1 to 10 or 0.1to 3 mg/kg. In one case, the IL-6 antibody or antibody portion thereofis administered in a formulation as a sterile aqueous solution having apH that ranges from about 5.0 to about 6.5 and comprising from about 1mg/ml to about 200 mg/ml of antibody, from about 1 millimolar to about100 millimolar of histidine buffer, from about 0.01 mg/ml to about 10mg/ml of polysorbate 80, from about 100 millimolar to about 400millimolar of trehalose, and from about 0.01 millimolar to about 1.0millimolar of disodium EDTA dihydrate. It is to be noted that dosagevalues may vary with the type and severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed compositions.

Another aspect provided herein are kits comprising an IL-6 antibody orantigen-binding portion or a composition comprising such an antibody orantigen-binding portion. A kit may include, in addition to the antibodyor composition, diagnostic or therapeutic agents. A kit can also includeinstructions for use in a diagnostic or therapeutic method. In one case,the kit includes the antibody or a composition comprising it and adiagnostic agent that can be used in a method described herein. Inanother case, the kit includes the antibody or a composition comprisingit and one or more therapeutic agents that can be used in a methoddescribed herein.

Diagnostic Methods of Use

Another aspect provided herein are diagnostic methods. The anti-IL-6antibodies or antigen binding portion thereof can be used to detect IL-6in a biological sample in vitro or in vivo. One aspect provides a methodfor diagnosing the presence or location of IL-6-expressing cells in asubject in need thereof, comprising the steps of injecting the antibodyinto the subject, determining the expression of IL-6 in the subject bylocalizing where the antibody has bound, comparing the expression in thesubject with that of a normal reference subject or standard, anddiagnosing the presence or location of the cells. The anti-IL-6antibodies may also be used as a marker for inflammation and/or for theinfiltration of immune cells, such as monocytes and T cells, into atissue.

The anti-IL-6 antibodies can be used in any suitable immunoassay,including, without limitation, an ELISA, a RIA, flow cytometry, tissueimmunohistochemistry, a Western blot or an immunoprecipitation. Theanti-IL-6 antibodies or antigen binding portion thereof can be used todetect IL-6 from humans. In another case, the anti-IL-6 antibodies canbe used to detect IL-6 from cynomolgus monkeys, rhesus monkeys androdents, such as mice and rats.

Methods for detecting IL-6 in a biological sample generally comprisecontacting the biological sample with an anti-IL-6 antibody or antigenbinding portions thereof and detecting the bound antibody. In one case,the anti-IL-6 antibody or antigen binding portion thereof is directlylabeled with a detectable label. In another case, the anti-IL-6 antibody(the first antibody) is unlabeled and a second antibody or othermolecule that can bind the anti-IL-6 antibody is labeled. A secondantibody is chosen that is able to specifically bind the particularspecies and class of the first antibody. For example, if the anti-IL-6antibody is a human IgG, then the secondary antibody could be ananti-human-IgG. Other molecules that can bind to antibodies include,without limitation, Protein A and Protein G, both of which are availablecommercially, e.g., from Pierce Chemical Co.

Suitable labels for the antibody or secondary antibody are disclosedherein and include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

IL-6 can also be assayed in a biological sample by a competitionimmunoassay utilizing IL-6 standards labeled with a detectable substanceand an unlabeled anti-IL-6 antibody. In this assay, the biologicalsample, the labeled IL-6 standards and the anti-IL-6 antibody arecombined and the amount of labeled IL-6 standard bound to the unlabeledantibody is determined. The amount of IL-6 in the biological sample isinversely proportional to the amount of labeled IL-6 standard bound tothe anti-IL-6 antibody.

One can use such immunoassays for a number of purposes. For example, theanti-IL-6 antibodies or antigen binding portions thereof can be used todetect IL-6 in cultured cells. In one case, the anti-IL-6 antibodies orantigen binding portions thereof are used to determine the amount ofIL-6 on the surface of cells that have been treated with variouscompounds. This method can be used to identify compounds that modulateIL-6 protein levels. According to this method, one sample of cells istreated with a test compound for a period of time while another sampleis left untreated. If the total IL-6 expression is to be measured, thecells are lysed and the total IL-6 expression is measured using anysuitable immunoassay. The total IL-6 expression in the treated versusthe untreated cells is compared to determine the effect of the testcompound.

Immunoassays for measuring total IL-6 expression include flow cytometryand immunohistochemistry. If the cell surface IL-6 expression is to bemeasured, the cells are not lysed, and the cell surface levels of IL-6are measured using one of the immunoassays described above. A preferredimmunoassay for determining cell surface levels of IL-6 includes thesteps of labeling the cell surface proteins with a detectable label,such as biotin or ¹²⁵I, immunoprecipitating the IL-6 with an anti-IL-6antibody and then detecting the labeled IL-6.

Another immunoassay for determining the localization of IL-6, e.g., cellsurface levels, is by using immunohistochemistry. An immunoassay todetect cell surface levels of IL-6 includes binding of an anti-IL-6antibody labeled with an appropriate fluorophore, such as fluorescein orphycoerythrin, and detecting the primary antibody using flow cytometry.In another example, the anti-IL-6 antibody is unlabeled and a secondantibody or other molecule that can bind the anti-IL-6 antibody islabeled. Methods such as ELISA, RIA, flow cytometry, Western blot,immunohistochemistry, cell surface labeling of integral membraneproteins and immunoprecipitation are well known in the art. See, e.g.,Harlow and Lane. In addition, the immunoassays can be scaled up for highthroughput screening in order to test a large number of compounds foreither activation or inhibitors of IL-6.

The anti-IL-6 antibodies or antigen binding portions thereof also can beused to determine the levels of IL-6 in a tissue or in cells derivedfrom the tissue. In some examples, the tissue is a diseased tissue or atissue biopsy. The tissue or biopsy can be used in an immunoassay todetermine, e.g., total IL-6 expression, cell surface levels of IL-6 orlocalization of IL-6 by the methods discussed above. Such methods can beused to determine whether a tissue expresses high levels of IL-6, whichcould be indicative that the tissue is a target for treatment withanti-IL-6 antibody.

IL-6 antibodies and antigen-binding portions thereof also can be used invivo to identify tissues and organs that express IL-6. In some cases,the anti-IL-6 antibodies are used to identify IL-6-expressing cells.Human anti-IL-6 antibodies may safely be used in vivo without elicitinga substantial immune response to the antibody upon administration,unlike antibodies of non-human origin or with humanized or chimericantibodies.

The method comprises the steps of administering a detectably labeledanti-IL-6 antibody or a composition comprising them to a patient in needof such a diagnostic test and subjecting the patient to imaging analysisto determine the location of the IL-6-expressing tissues. Imaginganalysis is well known in the medical art, and includes, withoutlimitation, x-ray analysis, magnetic resonance imaging (MRI) or computedtomography (CT). The antibody can be labeled with any agent suitable forin vivo imaging, for example a contrast agent, such as barium, which canbe used for x-ray analysis, or a magnetic contrast agent, such as agadolinium chelate, which can be used for MRI or CT. Other labelingagents include, without limitation, radioisotopes, such as ⁹⁹Tc. Inanother case, the anti-IL-6 antibody will be unlabeled and will beimaged by administering a second antibody or other molecule that isdetectable and that can bind the anti-IL-6 antibody. In one example, abiopsy is obtained from the patient to determine whether the tissue ofinterest expresses IL-6.

The detectably labeled anti-IL-6 may comprise a fluorophore. In certaincases, the fluorophore is selected from the group consisting of anear-infrared fluorescent dye, dinitrophenyl, fluorescein andderivatives thereof, rhodamine, derivatives of rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine, Texasred, Rhodamine green, Oregon green, Cascade blue, phycoerythrin, CY3,CY5, CY2, CY7, coumarin, infrared 40, MR 200, IRD 40, Alexa Fluor,Cascade Blue, Tetramethylrhodamine, Pacific Blue, SYBR, and BODIPY. Inanother example, the fluorophore includes one of the following compoundswith their emission maxima indicated in nm in parentheses, Cy2™ (506),GFP (Red Shifted) (507), YO-PRO®-1 (509), YOYO®-1 (509), Calcein (517),FITC (518), FluorX® (519), Alexa® (520), Rhodamine 110 (520), 5-FAM(522), Oregon Green® 500 (522), Oregon Green® 488 (524), RiboGreen®(525), Rhodamine Green® (527), Rhodamine 123 (529), Magnesium Green®(531), Calcium Green® (533), TO-PRO®-1 (533), TOTO®-1 (533), JOE (548),BODIPY® 530/550 (550), Dil (565), BODIPY® (568), BODIPY® 558/568 (568),BODIPY® 564/570 (570), Cy3® (570), Alexa® 546 (570), TRITC (572),Magnesium Orange® (575), Phycoerythrin R&B (575), Rhodamine Phalloidin(575), Calcium Orange® (576), Pyronin Y (580), Rhodamine B (580), TAMRA(582), Rhodamine Red® (590), Cy3.5® (596), ROX (608), Calcium Crimson™(615), Alexa® 594 (615), Texas Red® (615), Nile Red (628), YO-PRO®-3(631), YOYO®-3 (631), R-phycocyanin (642), C-Phycocyanin (648),TO-PRO®-3 (660), TOTO®-3 (660), DiD DilC(5) (665), Cy5™ (670),Thiadicarbocyanine (671) and Cy5.5 (694).

In yet a further example, the anti-IL-6 antibodies may also be used todetermine the reduction in surface cell expression of IL-6 on cells, forexample, lymphocytes or monocytes.

Human anti-IL-6 antibodies or antigen-binding portions thereof minimizethe immunogenic and allergic responses intrinsic to non-human ornon-human-derivatized monoclonal antibodies (Mabs), and thus increasethe efficacy and safety of the administered antibodies orantigen-binding portions thereof.

Another aspect provides human anti-IL-6 antibodies encoded in part by ahuman germline sequence. The V_(H), V_(K), V_(λ) genes are classifiedinto families on the basis of sequence homology. Two V_(H), V_(K), orV_(λ) genes belong to the same family if they share the same nucleotidesequence at more than 80% of the positions. An anti-IL-6 antibody maycomprise a human kappa light chain (V_(K)) or a human lambda light chain(V_(λ)) or an amino acid sequence derived therefrom. In some casescomprising a lambda light chain, the light chain variable domain (V_(L))is encoded in part by a human V_(λ)1, V_(λ)2, V_(λ)3, V_(λ)4, V_(λ)5,V_(λ)6, V_(λ)7, V_(λ)8, V_(λ)9, or V_(λ)10 family gene (Williams S. C.et al. J. Mol. Bio. 264:220-232, 1996). In some cases comprising a kappalight chain, the light chain variable domain (V_(L)) is encoded in partby a human V_(K)I, V_(K)II, V_(K)III, V_(K)IV, V_(K)V, or V_(K)VI familygene (Cox J. P. L., et al, Eur. J. Immunol 24:827-836, 1994), preferablya V_(K)I, V_(K)II, V_(K)III, or V_(K)IV family gene, and preferably aV_(K)I or V_(K)VI family gene. In some cases, the light chain germlinesequence is selected from human VK sequences including, but not limitedto, A1, A10, A11, A14, A17, A18, A19, A2, A20, A23, A26, A27, A3, A30,A5, A7, B2, B3, L1, L10, L11, L12, L14, L15, L16, L18, L19, L2, L20,L22, L23, L24, L25, L4/18a, L5, L6, L8, L9, O1, O11, O12, O14, O18, O2,O4, and O8. In certain cases, this light chain human germline frameworkis selected from V1-11, V1-13, V1-16, V1-17, V1-18, V1-19, V1-2, V1-20,V1-22, V1-3, V1-4, V1-5, V1-7, V1-9, V2-1, V2-11, V2-13, V2-14, V2-15,V2-17, V2-19, V2-6, V2-7, V2-8, V3-2, V3-3, V3-4, V4-1, V4-2, V4-3,V4-4, V4-6, V5-1, V5-2, V5-4, and V5-6. An anti-IL-6 antibody maycomprise a heavy chain variable domain (V_(H)) encoded by a humanV_(H)1, V_(H)2, V_(H)3, V_(H)4, V_(H)S, V_(H)6 or V_(H)7 family gene. Inparticular examples, this heavy chain human germline framework isselected from VH1-18, VH1-2, VH1-24, VH1-3, VH1-45, VH1-46, VH1-58,VH1-69, VH1-8, VH2-26, VH2-5, VH2-70, VH3-11, VH3-13, VH3-15, VH3-16,VH3-20, VH3-21, VH3-23, VH3-30, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48,VH3-49, VH3-53, VH3-64, VH3-66, VH3-7, VH3-72, VH3-73, VH3-74, VH3-9,VH4-28, VH4-31, VH4-34, VH4-39, VH4-4, VH4-59, VH4-61, VH5-51, VH6-1,and VH7-81. In particular cases, the light chain variable region and/orheavy chain variable region comprises a framework region or at least aportion of a framework region (e.g., containing 2 or 3 subregions, suchas FR2 and FR3). In certain cases, at least FRL1, FRL2, FRL3, or FRL4 isfully human. In other examples, at least FRH1, FRH2, FRH3, or FRH4 isfully human. In some cases, at least FRL1, FRL2, FRL3, or FRL4 is agermline sequence (e.g., human germline) or comprises human consensussequences for the particular framework (readily available at the sourcesof known human Ig sequences described herein). In other examples, atleast FRH1, FRH2, FRH3, or FRH4 is a germline sequence (e.g., humangermline) or comprises human consensus sequences for the particularframework.

The V_(L) of the IL-6 antibody may comprise one or more amino acidsubstitutions relative to the germline amino acid sequence of the humangene. In some cases, the V_(L) of the IL-6 antibody comprises 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 amino acid substitutions relative to thegermline amino acid sequence. In an example, one or more of thosesubstitutions from germline is in the CDR regions of the light chain. Inone example, the amino acid substitutions relative to germline are atone or more of the same positions as the substitutions relative togermline in any one or more of the V_(L) of antibodies 9C8 IgG1, 9C8IgG2, 9C8 N68T T83S IgG1, 9C8 N68T T83S IgG2, 9C8 E31G N68T T83S IgG1,9C8 E31G N68T T83S IgG2, 9C8 I24V N68T T83S IgG1, 9C8 I24V N68T T83SIgG2 and 22B5 IgG1. For example, the V_(L) of an IL-6 antibody maycontain one or more amino acid substitutions compared to germline foundin the V_(L) of antibody 9C8 IgG1. In some cases, the amino acid changesare at one or more of the same positions, but involve a differentsubstitution than in the reference antibody.

In some cases, amino acid changes relative to germline occur at one ormore of the same positions as in any of the V_(L) of antibodies 9C8IgG1, 9C8 IgG2, 9C8 N68T T83S IgG1, 9C8 N68T T83S IgG2, 9C8 E31G N68TT83S IgG1, 9C8 E31G N68T T83S IgG2, 9C8 I24V N68T T83S IgG1, 9C8 I24VN68T T83S IgG2 and 22B5 IgG1, but the changes may represent conservativeamino acid substitutions at such position(s) relative to the amino acidin the reference antibody. For example, if a particular position in oneof these antibodies is changed relative to germline and is glutamate,one may substitute aspartate at that position. Similarly, if an aminoacid substitution compared to germline is serine, one may conservativelysubstitute threonine for serine at that position.

In some cases, the light chain of the human anti-IL-6 antibody comprisesthe V_(L) amino acid sequence of antibody 9C8 IgG1, 9C8 IgG2, 9C8 N68TT83S IgG1, 9C8 N68T T83S IgG2, 9C8 E31G N68T T83S IgG1, 9C8 E31G N68TT83S IgG2, 9C8 I24V N68T T83S IgG1, 9C8 I24V N68T T83S IgG2 and 22B5IgG1 or the amino acid sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 conservative amino acid substitutions and/or a total of up to 3non-conservative amino acid substitutions. In some cases, the lightchain comprises the amino acid sequence from the beginning of the CDR1to the end of the CDR3 of any one of the foregoing antibodies.

In some cases, the light chain may comprise CDR1, CDR2 and CDR3 regionsindependently selected from the light chain CDR1, CDR2 and CDR3,respectively, of the light chain of antibodies 9C8 IgG1, 9C8 IgG2, 9C8N68T T83S IgG1, 9C8 N68T T83S IgG2, 9C8 E31G N68T T83S IgG1, 9C8 E31GN68T T83S IgG2, 9C8 I24V N68T T83S IgG1, 9C8 I24V N68T T83S IgG2 and22B5 IgG1, or CDR regions each having less than 4 or less than 3conservative amino acid substitutions and/or a total of three or fewernon-conservative amino acid substitutions. In some cases, the lightchain of the anti-IL-6 antibody comprises a light chain CDR1, CDR2, andCDR3, each of which are independently selected from the light chainCDR1, CDR2 and CDR3 regions of monoclonal antibody 9C8 IgG1, 9C8 IgG2,9C8 N68T T83S IgG1, 9C8 N68T T83S IgG2, 9C8 E31G N68T T83S IgG1, 9C8E31G N68T T83S IgG2, 9C8 I24V N68T T83S IgG1, 9C8 I24V N68T T83S IgG2and 22B5 IgG1. In certain cases, the light chain of the anti-IL-6antibody comprises the light chain CDR1, CDR2 and CDR3 regions of anantibody comprising the amino acid sequence of the V_(L) region of anantibody selected from 9C8 IgG1, 9C8 IgG2, 9C8 N68T T83S IgG1, 9C8 N68TT83S IgG2, 9C8 E31G N68T T83S IgG1, 9C8 E31G N68T T83S IgG2, 9C8 I24VN68T T83S IgG1, 9C8 I24V N68T T83S IgG2 and 22B5 IgG1 or the CDR regionseach having less than 4 or less than 3 conservative amino acidsubstitutions and/or a total of three or fewer non-conservative aminoacid substitutions.

In some cases, the variable domain (V_(H)) is encoded, at least in part,by a human gene. In some cases, the V_(H) sequence of the IL-6 antibodycontains one or more amino acid substitutions, deletions or insertions(additions) relative to the germline amino acid sequence. In some cases,the variable domain of the heavy chain comprises 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, or 17 mutations from the germline aminoacid sequence. In some cases, the mutation(s) are non-conservativesubstitutions, deletions or insertions, compared to the germline aminoacid sequence. In some examples, the mutations are in the CDR regions ofthe heavy chain. In some examples, the amino acid changes are made atone or more of the same positions as the mutations from germline in anyone or more of the V_(H) of antibodies 9C8 IgG1, 9C8 IgG2, 9C8 N68T T83SIgG1, 9C8 N68T T83S IgG2, 9C8 E31G N68T T83S IgG1, 9C8 E31G N68T T83SIgG2, 9C8 I24V N68T T83S IgG1, 9C8 I24V N68T T83S IgG2 and 22B5 IgG1. Inother examples, the amino acid changes are at one or more of the samepositions but involve a different mutation than in the referenceantibody.

In some cases, the heavy chain comprises the V_(H) amino acid sequenceof antibody 9C8 IgG1, 9C8 IgG2, 9C8 N68T T83S IgG1, 9C8 N68T T83S IgG2,9C8 E31G N68T T83S IgG1, 9C8 E31G N68T T83S IgG2, 9C8 I24V N68T T83SIgG1, 9C8 I24V N68T T83S IgG2 and 22B5 IgG1 the V_(H) amino acidsequence having up to 1, 2, 3, 4, 6, 8, or 10 conservative amino acidsubstitutions and/or a total of up to 3 non-conservative amino acidsubstitutions. In some examples, the heavy chain comprises the aminoacid sequence from the beginning of the CDR1 to the end of the CDR3 ofany one of the foregoing antibodies.

In some cases, the heavy chain comprises the heavy chain CDR1, CDR2 andCDR3 regions of antibody 9C8 IgG1, 9C8 IgG2, 9C8 N68T T83S IgG1, 9C8N68T T83S IgG2, 9C8 E31G N68T T83S IgG1, 9C8 E31G N68T T83S IgG2, 9C8I24V N68T T83S IgG1, 9C8 I24V N68T T83S IgG2 and 22B5 IgG1 or the CDRregions each having less than 8, less than 6, less than 4, or less than3 conservative amino acid substitutions and/or a total of three or fewernon-conservative amino acid substitutions.

In some cases, the heavy chain CDR regions are independently selectedfrom the CDR regions of antibodies 9C8 IgG1, 9C8 IgG2, 9C8 N68T T83SIgG1, 9C8 N68T T83S IgG2, 9C8 E31G N68T T83S IgG1, 9C8 E31G N68T T83SIgG2, 9C8 I24V N68T T83S IgG1, 9C8 I24V N68T T83S IgG2 and 22B5 IgG1. Inother cases, the heavy chain comprises CDR regions independentlyselected from two or more V_(H) regions selected from 9C8 IgG1, 9C8IgG2, 9C8 N68T T83S IgG1, 9C8 N68T T83S IgG2, 9C8 E31G N68T T83S IgG1,9C8 E31G N68T T83S IgG2, 9C8 I24V N68T T83S IgG1, 9C8 I24V N68T T83SIgG2 and 22B5 IgG1.

In other cases, the antibody comprises a light chain and a heavy chain.In a further example, the light chain CDRs and the heavy chain CDRs arefrom the same antibody.

One type of amino acid substitution that may be made is to change one ormore cysteines in the antibody, which may be chemically reactive, toanother residue, such as, without limitation, alanine or serine. In oneexample, there is a substitution of a non-canonical cysteine. Thesubstitution can be made in a CDR or framework region of a variabledomain or in the constant domain of an antibody. In some cases, thecysteine is canonical.

Another type of amino acid substitution that may be made is to changeany potential proteolytic sites in the antibody. Such sites may occur ina CDR or framework region of a variable domain or in the constant domainof an antibody. Substitution of cysteine residues and removal ofproteolytic sites may decrease the risk of any heterogeneity in theantibody product and thus increase its homogeneity. Another type ofamino acid substitution is to eliminate asparagine-glycine pairs, whichform potential deamidation sites, by altering one or both of theresidues.

In certain cases, the heavy and light chains of the IL-6 antibodies mayoptionally include a signal sequence.

In some cases, the antibody comprises the heavy and light chain variantsof monoclonal antibodies 9C8 IgG1, 9C8 IgG2, 9C8 N68T T83S IgG1, 9C8N68T T83S IgG2, 9C8 E31G N68T T83S IgG1, 9C8 E31G N68T T83S IgG2, 9C8I24V N68T T83S IgG1, 9C8 I24V N68T T83S IgG2 and 22B5 IgG1. As discussedin greater detail in EXAMPLE 3, numerous heavy and light chain variantmutations were made to match those in the germline CDR regions. Thespecific amino acids that were mutated to arrive at the germlineversions are apparent to those of skill in the art by comparing thesequences of the germline vs. a non-germline antibody. For example, oneamino acid substitution is provided in the heavy chain of antibody 9C8,wherein an isoleucine at residue 24 is changed to a valine and isreferred to as 9C8 I24V. A second exemplary amino acid substitution isin the light chain of antibody 9C8, and substitutes the lysine atresidue 92 with an asparagine and is referred to as 9C8 K92N.

As will be appreciated, gene utilization analysis provides only alimited overview of antibody structure. As human B-cells stochasticallygenerate V-D-J heavy or V-J kappa light chain transcripts, there are anumber of secondary processes that occur, including, without limitation,somatic hypermutation, additions, and CDR3 extensions. Accordingly, tofurther examine antibody structures, predicted amino acid sequences ofthe antibodies were generated from the cDNAs obtained from the clones.In addition, N-terminal amino acid sequences were obtained throughprotein sequencing.

Class and Subclass of Anti-IL-6 Antibodies

The class (e.g., IgG, IgM, IgE, IgA, or IgD) and subclass (e.g. IgG1,IgG2, IgG3, or IgG4) of IL-6 antibodies may be determined by anysuitable method. In general, the class and subclass of an antibody maybe determined using antibodies that are specific for a particular classand subclass of antibody. Such antibodies are commercially available.The class and subclass can be determined by ELISA, or Western Blot, aswell as other techniques.

Alternatively, the class and subclass may be determined by sequencingall or a portion of the constant domains of the heavy and/or lightchains of the antibodies, comparing their amino acid sequences to theknown amino acid sequences of various class and subclasses ofimmunoglobulins, and determining the class and subclass of theantibodies. The IL-6 antibodies can be an IgG, an IgM, an IgE, an IgA,or an IgD molecule. For example, the IL-6 antibodies can be an IgG thatis an IgG1, IgG2, IgG3, or an IgG4 subclass. In one example, the IL-6antibodies are IgG2 subclass. In another example, the IL-6 antibodiesare IgG1 subclass.

In one aspect methods are provided for converting the class or subclassof an IL-6 antibody to another class or subclass. In some cases, anucleic acid molecule encoding a V_(L) or V_(H) that does not includesequences encoding C_(L) or C_(H) is isolated using any suitablemethods. The nucleic acid molecule then is operatively linked to anucleic acid sequence encoding a C_(L) or C_(H) from a desiredimmunoglobulin class or subclass. This can be achieved using a vector ornucleic acid molecule that comprises a C_(L) or C_(H) chain, asdescribed above. For example, an IL-6 antibody that was originally IgMcan be class switched to an IgG. Further, the class switching may beused to convert one IgG subclass to another, e.g., from IgG1 to IgG2.Another method for producing an antibody comprising a desired isotypecomprises the steps of isolating a nucleic acid encoding a heavy chainof an IL-6 antibody and a nucleic acid encoding a light chain of an IL-6antibody, isolating the sequence encoding the V_(H) region, ligating theV_(H) sequence to a sequence encoding a heavy chain constant domain ofthe desired isotype, expressing the light chain gene and the heavy chainconstruct in a cell, and collecting the IL-6 antibody with the desiredisotype.

Binding Affinity of IL-6 Antibodies to IL-6

The binding affinity and dissociation rate of an anti-IL-6 antibody toIL-6 can be determined by any suitable method. The binding affinity canbe measured by ELISAs, RIAs, flow cytometry, and surface plasmonresonance, such as BIACORE™. The dissociate rate can be measured bysurface plasmon resonance. One can determine whether an antibody hassubstantially the same K_(D) as an anti-IL-6 antibody by using anysuitable method. Example 7 exemplifies a method for determining affinityconstants of anti-IL-6 monoclonal antibodies.

Identification of IL-6 Epitopes Recognized by Anti-IL-6 Antibodies

One can determine whether an antibody binds to the same epitope orcross-competes for binding with an IL-6 antibody by using any suitablemethod. In one example, one allows an IL-6 antibody to bind to IL-6under saturating conditions and then measures the ability of the testantibody to bind to IL-6. If the test antibody is able to bind to IL-6at the same time as the IL-6 antibody, then the test antibody binds to adifferent epitope as the IL-6 antibody. However, if the test antibody isnot able to bind to IL-6 at the same time, then the test antibody bindsto the same epitope, an overlapping epitope, or an epitope that is inclose proximity to the epitope bound by the human IL-6 antibody. Thisexperiment can be performed using an ELISA, a RIA, BIACORE™, or flowcytometry (FACS).

To test whether an IL-6 antibody cross-competes with another IL-6antibody, one may use the competition method described herein in twodirections, i.e., determining if the reference antibody blocks the testantibody and vice versa. In one example, the experiment is performedusing an ELISA.

Methods of Producing Antibodies

IL-6 antibodies or antigen-binding portions thereof can be produced by avariety of techniques, including conventional monoclonal antibodymethodology, for example the standard somatic cell hybridizationtechnique of Kohler and Milstein (1975) Nature 256: 495. Othertechniques for producing monoclonal antibodies can also be employed suchas viral or oncogenic transformation of B lymphocytes.

An exemplary animal system for preparing hybridomas is a murine system.Immunization protocols and techniques for isolation of immunizedsplenocytes for fusion are known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies can be prepared based on the sequenceof a murine monoclonal antibody prepared as described above. DNAencoding the heavy and light chain immunoglobulins can be obtained fromthe murine hybridoma of interest and engineered to contain non-murine(e.g., human) immunoglobulin sequences using standard molecular biologytechniques. For example, to create a chimeric antibody, the murinevariable regions can be linked to human constant regions using methodsknown in the art (see e.g., U.S. Pat. No. 4,816,567 to Cabilly et al.).To create a humanized antibody, the murine CDR regions can be insertedinto a human framework using methods known in the art (see e.g., U.S.Pat. No. 5,225,539, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762;and 6,180,370).

In one case, the anti-IL-6 antibodies are human monoclonal antibodies.Such human monoclonal antibodies directed against IL-6 can be generatedusing transgenic or transchromosomic mice carrying parts of the humanimmune system rather than the mouse system. These transgenic andtranschromosomic mice include mice referred to herein as the HuMAbMouse® and KM Mouse®, respectively, and are collectively referred toherein as “human Ig mice.”

The HuMAb Mouse® (Medarex®, Inc.) contains human immunoglobulin geneminiloci that encode unrearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg, et al.(1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal (Lonberg, N. et al. (1994); reviewed in Lonberg, N. (1994)Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding, F. andLonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546). Preparation anduse of the HuMAb Mouse®, and the genomic modifications carried by suchmice, is further described in Taylor, L. et al. (1992) Nucleic AcidsResearch 20:6287-6295; Chen, J. et al. (1993) International Immunology5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. etal. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol.152:2912-2920; Taylor, L. et al. (1994) International Immunology 6:579-591; and Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851, the contents of all of which are hereby specificallyincorporated by reference in their entirety. See further, U.S. Pat. Nos.5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397;5,661,016; 5,814,318; 5,874,299; and 5,770,429; U.S. Pat. No. 5,545,807;PCT Publication Nos.: WO 92/03918, WO 93/12227, WO 94/25585, WO97/13852, WO 98/24884 and WO 99/45962; and PCT Publication No. WO01/14424.

In another aspect, human anti-IL-6 antibodies can be raised using amouse that carries human immunoglobulin sequences on transgenes andtranschomosomes, such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. Such mice, referredto herein as “KM Mice™”, are described in detail in PCT Publication No.WO 02/43478.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseIL-6 antibodies. For example, an alternative transgenic system referredto as the Xenomouse™ (Abgenix, Inc.) can be used; such mice aredescribed in, for example, U.S. Pat. Nos. 5,939,598; 6,075,181;6,114,598; 6,150,584; and 6,162,963.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseIL-6 antibodies. For example, mice carrying both a human heavy chaintranschromosome and a human light chain tranchromosome, referred to as“TC mice” can be used; such mice are described in Tomizuka et al. (2000)Proc. Natl. Acad. Sci. USA 97:722-727. Furthermore, cows carrying humanheavy and light chain transchromosomes have been described in the art(Kuroiwa et al. (2002) Nature Biotechnology 20:889-894) and can be usedto raise IL-6 antibodies.

Human monoclonal antibodies can also be prepared using SCID mice intowhich human immune cells have been reconstituted such that a humanantibody response can be generated upon immunization. Such mice aredescribed in, for example, U.S. Pat. Nos. 5,476,996; and 5,698,767.

Immunization of Human Ig Mice

Production of Antibodies and Antibody-Producing Cell Lines

After immunization of an animal with an IL-6 antigen, antibodies and/orantibody-producing cells can be obtained from the animal. In some cases,IL-6 antibody-containing serum is obtained from the animal by bleedingor sacrificing the animal. The serum may be used as it is obtained fromthe animal, an immunoglobulin fraction may be obtained from the serum,or the IL-6 antibodies may be purified from the serum.

In some cases, antibody-producing immortalized cell lines are preparedfrom cells isolated from the immunized animal. After immunization, theanimal is sacrificed and lymph node and/or splenic B cells areimmortalized by any means known in the art. Methods of immortalizingcells include, but are not limited to, transfecting them with oncogenes,infecting them with an oncogenic virus and cultivating them underconditions that select for immortalized cells, subjecting them tocarcinogenic or mutating compounds, fusing them with an immortalizedcell, e.g., a myeloma cell, and inactivating a tumor suppressor gene. Iffusion with myeloma cells is used, the myeloma cells preferably do notsecrete immunoglobulin polypeptides (a non-secretory cell line).Immortalized cells are screened using IL-6, a portion thereof, or a cellexpressing IL-6. In some cases, the initial screening is performed usingan enzyme-linked immunoassay (ELISA) or a radioimmunoassay. An exampleof ELISA screening is provided in PCT Publication No. WO 00/37504,incorporated herein by reference.

IL-6 antibody-producing cells, e.g., hybridomas, are selected, clonedand further screened for desirable characteristics, including robustgrowth, high antibody production and desirable antibody characteristics.Hybridomas can be expanded in vivo in syngeneic animals, in animals thatlack an immune system, e.g., nude mice, or in cell culture in vitro.Methods of selecting, cloning and expanding hybridomas are well known tothose of ordinary skill in the art.

In one example, the immunized animal is a non-human animal thatexpresses human immunoglobulin genes and the splenic B cells are fusedto a myeloma cell line from the same species as the non-human animal.One such immunized animal is a Kirin TC Mouse™ mouse and the myelomacell line is a non-secretory mouse myeloma. In a further example themyeloma cell line is Sp2/0-Ag14 (American Type Culture Collection (ATCC)CRL-1581).

Also provided are methods for producing a cell line that produces ahuman monoclonal antibody or a antigen-binding portion thereof directedto IL-6 comprising: (a) immunizing a non-human transgenic animaldescribed herein with IL-6, a portion of IL-6 or a cell or tissueexpressing Il-6; (b) allowing the transgenic animal to mount an immuneresponse to IL-6; (c) isolating antibody-producing cells from thetransgenic animal; (d) immortalizing the antibody-producing cells; (e)creating individual monoclonal populations of the immortalizedantibody-producing cells; and (f) screening the immortalizedantibody-producing cells to identify an antibody directed to IL-6.

In another aspect, hybridomas are provided that produce a human IL-6antibody. The human IL-6 antibody produced by the hybridoma may be anantagonist of IL-6. The hybridomas may be produced in a non-human,non-mouse species such as, for example, rats, sheep, pigs, goats, cattleor horses.

In one case, antibody-producing cells are isolated and expressed in ahost cell, for example myeloma cells. In still another example, atransgenic animal is immunized with IL-6, primary cells (e.g., spleen orperipheral blood cells) are isolated from an immunized transgenic animaland individual cells producing antibodies specific for the desiredantigen are identified. Polyadenylated mRNA from each individual cell isisolated and reverse transcription polymerase chain reaction (RT-PCR) isperformed using sense primers that anneal to variable region sequences,e.g., degenerate primers that recognize most or all of the FR1 regionsof human heavy and light chain variable region genes and anti-senseprimers that anneal to constant or joining region sequences. cDNAs ofthe heavy and light chain variable domains are then cloned and expressedin any suitable host cell, e.g., a myeloma cell, as chimeric antibodieswith respective immunoglobulin constant regions, such as the heavy chainand κ or λ constant domains. See Babcook, J. S. et al. (1996) Proc.Natl. Acad. Sci. USA 93: 7843-48, incorporated herein by reference. IL-6antibodies may then be identified and isolated.

Recombinant Methods of Producing Antibodies

IL-6 antibodies or antigen-binding portions thereof can be prepared byrecombinant expression of immunoglobulin light and heavy chain genes ina host cell. For example, to express an antibody recombinantly, a hostcell is transfected with one or more recombinant expression vectorscarrying DNA fragments encoding the immunoglobulin light and heavychains of the antibody such that the light and heavy chains areexpressed in the host cell and, preferably, secreted into the medium inwhich the host cells are cultured, from which medium the antibodies canbe recovered. Various recombinant DNA methodologies are used to obtainantibody heavy and light chain genes, to incorporate these genes intorecombinant expression vectors and to introduce the vectors into hostcells, such as those described in Sambrook, Fritsch and Maniatis (eds),Molecular Cloning; A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), Ausubel, F. M. et al. (eds.) Current Protocols inMolecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat.No. 4,816,397, the disclosures of which are incorporated herein byreference.

Mutations and Modifications

To express the IL-6 antibodies, DNA fragments encoding V_(H) and V_(L)regions can first be obtained using any of the methods discussed herein.Various mutations, deletions, and/or additions can also be introducedinto the DNA sequences using various suitable methods. For example,mutagenesis can be carried out using standard methods, such asPCR-mediated mutagenesis, in which the mutated nucleotides areincorporated into the PCR primers such that the PCR product contains thedesired mutations or site-directed mutagenesis. One type ofsubstitution, for example, that may be made is to change one or morecysteines in the antibody, which may be chemically reactive, to anotherresidue, such as, without limitation, alanine or serine. For example,there can be a substitution of a non-canonical or canonical cysteine.The substitution can be made in a CDR or framework region of a variabledomain or in the constant domain of an antibody. The antibodies may alsobe mutated in the variable domains of the heavy and/or light chains,e.g., to alter a binding property of the antibody. For example, amutation may be made in one or more of the CDR regions to increase ordecrease the K_(D) of the antibody for IL-6, to increase or decreasek_(off), or to alter the binding specificity of the antibody. Techniquesin site-directed mutagenesis include, for example, Sambrook et al. andAusubel et al., which are incorporated herein by reference.

A mutation may also be made in a framework region or constant domain toincrease the half-life of an IL-6 antibody. See, e.g., PCT PublicationNo. WO 00/09560, incorporated herein by reference. A mutation in aframework region or constant domain can also be made to alter theimmunogenicity of the antibody, to provide a site for covalent ornon-covalent binding to another molecule, or to alter such properties ascomplement fixation, FcR binding and antibody-dependent cell-mediatedcytotoxicity (ADCC). A single antibody may have mutations in any one ormore of the CDRs or framework regions of the variable domain or in theconstant domain.

In a process known as “germlining”, certain amino acids in the V_(H) andV_(L) sequences can be mutated to match those found naturally ingermline V_(H) and V_(L) sequences. In particular, the amino acidsequences of the framework regions in the V_(H) and V_(L) sequences canbe mutated to match the germline sequences to reduce the risk ofimmunogenicity when the antibody is administered. Germline DNA sequencesfor human V_(H) and V_(L) genes are known in the art (see e.g., the“Vbase” human germline sequence database; see also Kabat, E. A., et al.(1991) Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242; Tomlinson et al. (1992) J. Mol. Biol. 227:776-798; and Cox etal. Eur. J. Immunol. 24:827-836 (1994); the contents of each of whichare incorporated herein by reference).

Another type of amino acid substitution that may be made is to removepotential proteolytic sites in the antibody. Such sites may occur in aCDR or framework region of a variable domain or in the constant domainof an antibody. Substitution of cysteine residues and removal ofproteolytic sites may decrease the risk of heterogeneity in the antibodyproduct and thus increase its homogeneity. Another type of amino acidsubstitution is to eliminate asparagine-glycine pairs, which formpotential deamidation sites, by altering one or both of the residues. Inanother example, the C-terminal lysine of the heavy chain of an IL-6antibody can be cleaved. In various examples, the heavy and light chainsof the IL-6 antibodies may optionally include a signal sequence. In somecases, the C-terminal lysine of the heavy chain of the anti-IL-6antibody may be proteolytically cleaved.

Once DNA fragments encoding the V_(H) and V_(L) segments are obtained,these DNA fragments can be further manipulated by standard recombinantDNA techniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes, or to a scFvgene. In these manipulations, a V_(L)- or V_(H)-encoding DNA fragment isoperatively linked to another DNA fragment encoding another protein,such as an antibody constant region or a flexible linker. The term“operatively linked”, as used in this context, is intended to mean thatthe two DNA fragments are joined such that the amino acid sequencesencoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the V_(H)-encodingDNA to another DNA molecule encoding heavy chain constant regions (CH1,CH2 and CH3). The sequences of human heavy chain constant region genesare known in the art (see e.g., Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably isan IgG1 or IgG2 constant region. The IgG1 constant region sequence canbe any of the various alleles or allotypes known to occur amongdifferent individuals, such as Gm(1), Gm(2), Gm(3), and Gm(17). Theseallotypes represent naturally occurring amino acid substitution in theIgG1 constant regions. For a Fab fragment heavy chain gene, theV_(H)-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region. The CH1 heavy chainconstant region may be derived from any of the heavy chain genes.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, C_(L). The sequences of humanlight chain constant region genes are known in the art (see e.g., Kabat,E. A., et al. (1991) Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242) and DNA fragments encompassing these regionscan be obtained by standard PCR amplification. The light chain constantregion can be a kappa or lambda constant region. The kappa constantregion may be any of the various alleles known to occur among differentindividuals, such as Inv(1), Inv(2), and Inv(3). The lambda constantregion may be derived from any of the three lambda genes.

To create a scFv gene, the V_(H)- and V_(L)-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the V_(H) andV_(L) sequences can be expressed as a contiguous single-chain protein,with the V_(L) and V_(H) regions joined by the flexible linker (seee.g., Bird et al. Science 242:423-426 (1988); Huston et al. Proc. Natl.Acad. Sci. USA 85:5879-5883 (1988); McCafferty et al., Nature348:552-554 (1990)). The single chain antibody may be monovalent, ifonly a single V_(H) and V_(L) are used, bivalent, if two V_(H) and V_(L)are used, or polyvalent, if more than two V_(H) and V_(L) are used.Bispecific or polyvalent antibodies may be generated that bindspecifically to IL-6 and to another molecule.

In another case, a fusion antibody may be made that comprises all or aportion of an IL-6 antibody linked to another polypeptide. In anothercase, only the variable domains of the IL-6 antibody are linked to thepolypeptide. In another case, the V_(H) domain of an IL-6 antibody islinked to a first polypeptide, while the V_(L) domain of an IL-6antibody is linked to a second polypeptide that associates with thefirst polypeptide in a manner such that the V_(H) and V_(L) domains caninteract with one another to form an antigen binding site. In anothercase, the V_(H) domain is separated from the V_(L) domain by a linkersuch that the V_(H) and V_(L) domains can interact with one another. TheV_(H)-linker-V_(L) antibody is then linked to the polypeptide ofinterest. In addition, fusion antibodies can be created in which two (ormore) single-chain antibodies are linked to one another. This is usefulif one wants to create a divalent or polyvalent antibody on a singlepolypeptide chain, or if one wants to create a bispecific antibody.

In other cases, other modified antibodies may be prepared using IL-6antibody encoding nucleic acid molecules. For instance, “Kappa bodies”(Ill et al., Protein Eng. 10: 949-57 (1997)), “Minibodies” (Martin etal., EMBO J. 13: 5303-9 (1994)), “Diabodies” (Holliger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993)), or “Janusins” (Traunecker etal., EMBO J. 10:3655-3659 (1991) and Traunecker et al., Int. J. Cancer(Suppl.) 7:51-52 (1992)) may be prepared using suitable molecularbiological techniques following the teachings discussed herein.

Bispecific antibodies or antigen-binding fragments can be produced by avariety of methods including fusion of hybridomas or linking of Fab′fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol.79:315-321 (1990), Kostelny et al., J. Immunol. 148:1547-1553 (1992). Inaddition, bispecific antibodies may be formed as “diabodies” or“Janusins.” In some cases, the bispecific antibody binds to twodifferent epitopes of IL-6. In some cases, the modified antibodiesdescribed herein are prepared using one or more of the variable domainsor CDR regions from a human IL-6 antibody.

Vectors and Host Cells

To express IL-6 antibodies and antigen-binding portions thereof, DNAsencoding partial or full-length light and heavy chains, obtained asdescribed herein, are inserted into expression vectors such that thegenes are operatively linked to transcriptional and translationalcontrol sequences. In this context, the term “operatively linked” isintended to mean that an antibody gene is ligated into a vector suchthat transcriptional and translational control sequences within thevector serve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. Expression vectors include, for example, plasmids,retroviruses, adenoviruses, adeno-associated viruses (AAV), plantviruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids,YACs, and EBV derived episomes. The antibody gene is ligated into avector such that transcriptional and translational control sequenceswithin the vector serve their intended function of regulating thetranscription and translation of the antibody gene. The expressionvector and expression control sequences are chosen to be compatible withthe expression host cell used. The antibody light chain gene and theantibody heavy chain gene can be inserted into separate vectors or bothgenes are inserted into the same expression vector. The antibody genesare inserted into the expression vector by various methods (e.g.,ligation of complementary restriction sites on the antibody genefragment and vector, or blunt end ligation if no restriction sites arepresent).

A convenient vector is one that encodes a functionally complete humanC_(H) or C_(L) immunoglobulin sequence, with appropriate restrictionsites engineered so that any V_(H) or V_(L) sequence can easily beinserted and expressed, as described above. In such vectors, splicingusually occurs between the splice donor site in the inserted J regionand the splice acceptor site preceding the human C domain, and also atthe splice regions that occur within the human C_(H) exons.Polyadenylation and transcription termination occur at nativechromosomal sites downstream of the coding regions. The recombinantexpression vector also can encode a signal peptide that facilitatessecretion of the antibody chain from a host cell. The antibody chaingene may be cloned into the vector such that the signal peptide islinked in-frame to the amino terminus of the immunoglobulin chain. Thesignal peptide can be an immunoglobulin signal peptide or a heterologoussignal peptide (i.e., a signal peptide from a non-immunoglobulinprotein).

In addition to the antibody chain genes, the recombinant expressionvectors carry regulatory sequences that control the expression of theantibody chain genes in a host cell. The design of the expressionvector, including the selection of regulatory sequences may depend onsuch factors as the choice of the host cell to be transformed, the levelof expression of protein desired. Regulatory sequences for mammalianhost cell expression include viral elements that direct high levels ofprotein expression in mammalian cells, such as promoters and/orenhancers derived from retroviral LTRs, cytomegalovirus (CMV) (such asthe CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40promoter/enhancer), adenovirus, (e.g., the adenovirus major latepromoter (AdMLP)), polyoma and strong mammalian promoters such as nativeimmunoglobulin and actin promoters. For further description of viralregulatory elements, and sequences thereof, see e.g., U.S. Pat. No.5,168,062, U.S. Pat. No. 4,510,245 and U.S. Pat. No. 4,968,615,incorporated herein by reference. Methods for expressing antibodies inplants, including a description of promoters and vectors, as well astransformation of plants is known in the art. See, e.g., U.S. Pat. No.6,517,529, incorporated herein by reference.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors may carry additional sequences, such assequences that regulate replication of the vector in host cells (e.g.,origins of replication) and selectable marker genes. The selectablemarker gene facilitates selection of host cells into which the vectorhas been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and5,179,017, incorporated herein by reference). For example, typically theselectable marker gene confers resistance to drugs, such as G418,hygromycin or methotrexate, on a host cell into which the vector hasbeen introduced. Selectable marker genes include the dihydrofolatereductase (DHFR) gene (for use in dhfr-host cells with methotrexateselection/amplification), the neomycin phosphotransferase gene (for G418selection), and the glutamate synthetase gene.

Nucleic acid molecules encoding IL-6 antibodies and vectors comprisingthese nucleic acid molecules can be used for transfection of a suitablemammalian, plant, bacterial or yeast host cell. Transformation can beany suitable method for introducing polynucleotides into a host cell.Methods for introduction of heterologous polynucleotides into mammaliancells include dextran-mediated transfection, calcium phosphateprecipitation, polybrene-mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,and direct microinjection of the DNA into nuclei. In addition, nucleicacid molecules may be introduced into mammalian cells by viral vectors.Methods of transforming cells are well known in the art. See, e.g., U.S.Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455, incorporatedherein by reference). Methods of transforming plant cells are well knownin the art, including, e.g., Agrobacterium-mediated transformation,biolistic transformation, direct injection, electroporation and viraltransformation. Methods of transforming bacterial and yeast cells arealso well known in the art.

Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC). These include, for example,Chinese hamster ovary (CHO) cells, NSO cells, SP2 cells, HEK-293T cells,NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, Africangreen monkey kidney cells (COS), human hepatocellular carcinoma cells(e.g., Hep G2), A549 cells, and a number of other cell lines. Cell linesof particular preference are selected through determining which celllines have high expression levels. Other cell lines that may be used areinsect cell lines, such as Sf9 or Sf21 cells. When recombinantexpression vectors encoding antibody genes are introduced into mammalianhost cells, the antibodies are produced by culturing the host cells fora period of time sufficient to allow for expression of the antibody inthe host cells or, more preferably, secretion of the antibody into theculture medium in which the host cells are grown. Antibodies can berecovered from the culture medium using various protein purificationmethods. Bacterial host cells include E. coli and Streptomyces species.Yeast host cells include Schizosaccharomyces pombe, Saccharomycescerevisiae and Pichia pastoris.

Further, expression of antibodies from production cell lines can beenhanced using any suitable techniques. For example, the glutaminesynthetase (the GS system) and DHFR gene expression systems are commonapproaches for enhancing expression under certain conditions. Highexpressing cell clones can be identified using conventional techniques,such as limited dilution cloning and Microdrop technology. The GS systemis discussed in European Patent Nos. 0 216 846, 0 256 055, 0 323 997 and0 338 841.

It is likely that antibodies expressed by different cell lines or intransgenic animals will have different glycosylation from each other.However, all antibodies encoded by the nucleic acid molecules providedherein, or comprising the amino acid sequences provided herein are partof the present disclosure, regardless of the glycosylation of theantibodies.

Phage Display Libraries

Also provided are methods for producing an IL-6 antibody orantigen-binding portion thereof comprising the steps of synthesizing alibrary of human antibodies on phage, screening the library with IL-6 ora portion thereof, isolating phage that bind IL-6, and obtaining theantibody from the phage. By way of example, one method for preparing thelibrary of antibodies for use in phage display techniques comprises thesteps of immunizing a non-human animal comprising human immunoglobulinloci with IL-6 or an antigenic portion thereof to create an immuneresponse, extracting antibody-producing cells from the immunized animal;isolating RNA encoding heavy and light chains of antibodies from theextracted cells, reverse transcribing the RNA to produce cDNA,amplifying the cDNA using primers, and inserting the cDNA into a phagedisplay vector such that antibodies are expressed on the phage.Recombinant IL-6 antibodies may be obtained in this way.

Recombinant IL-6 human antibodies can be isolated by screening arecombinant combinatorial antibody library. Preferably the library is ascFv phage display library, generated using human V_(L) and V_(H) cDNAsprepared from mRNA isolated from B cells. Methods for preparing andscreening such libraries are known in the art. Kits for generating phagedisplay libraries are commercially available (e.g., the PharmaciaRecombinant Phage Antibody System, catalog no. 27-9400-01; and theStratagene SurfZAP™ phage display kit, catalog no. 240612). There alsoare other methods and reagents that can be used in generating andscreening antibody display libraries (see, e.g., U.S. Pat. No.5,223,409; PCT Publication Nos. WO 92/18619, WO 91/17271, WO 92/20791,WO 92/15679, WO 93/01288, WO 92/01047, WO 92/09690; Fuchs et al.,Bio/Technology 9:1370-1372 (1991); Hay et al., Hum. Antibod. Hybridomas3:81-85 (1992); Huse et al., Science 246:1275-1281 (1989); McCafferty etal., Nature 348:552-554 (1990); Griffiths et al., EMBO J. 12:725-734(1993); Hawkins et al., J. Mol. Biol. 226:889-896 (1992); Clackson etal., Nature 352:624-628 (1991); Gram et al., Proc. Natl. Acad. Sci. USA89:3576-3580 (1992); Garrad et al., Bio/Technology 9:1373-1377 (1991);Hoogenboom et al., Nuc. Acid Res. 19:4133-4137 (1991); and Barbas etal., Proc. Natl. Acad. Sci. USA 88:7978-7982 (1991), all incorporatedherein by reference.

To isolate and produce human IL-6 antibodies with the desiredcharacteristics, a human IL-6 antibody is first used to select humanheavy and light chain sequences having similar binding activity towardIL-6, using the epitope imprinting methods described in PCT PublicationNo. WO 93/06213, incorporated herein by reference. The antibodylibraries used in this method are preferably scFv libraries prepared andscreened as described in PCT Publication No. WO 92/01047, McCafferty etal., Nature 348:552-554 (1990); and Griffiths et al., EMBO J. 12:725-734(1993), all incorporated herein by reference.

Once initial human V_(L) and V_(H) domains are selected, “mix and match”experiments are performed, in which different pairs of the initiallyselected V_(L) and V_(H) segments are screened for IL-6 binding toselect preferred V_(L)/V_(H) pair combinations. Additionally, to furtherimprove the quality of the antibody, the V_(L) and V_(H) segments of thepreferred V_(L)/V_(H) pair(s) can be randomly mutated, preferably withinthe CDR3 region of V_(H) and/or V_(L), in a process analogous to the invivo somatic mutation process responsible for affinity maturation ofantibodies during a natural immune response. This in vitro affinitymaturation can be accomplished by amplifying V_(H) and V_(L) domainsusing PCR primers complimentary to the V_(H) CDR3 or V_(L) CDR3,respectively, which primers have been “spiked” with a random mixture ofthe four nucleotide bases at certain positions such that the resultantPCR products encode V_(H) and V_(L) segments into which random mutationshave been introduced into the V_(H) and/or V_(L) CDR3 regions. Theserandomly mutated V_(H) and V_(L) segments can be re-screened for bindingto IL-6.

Following screening and isolation of an IL-6 antibody from a recombinantimmunoglobulin display library, nucleic acids encoding the selectedantibody can be recovered from the display package (e.g., from the phagegenome) and subcloned into other expression vectors by standardrecombinant DNA techniques. If desired, the nucleic acid can further bemanipulated to create other antibody forms, as described herein. Toexpress a recombinant human antibody isolated by screening of acombinatorial library, the DNA encoding the antibody is cloned into arecombinant expression vector and introduced into mammalian host cells,as described above.

Deimmunized Antibodies

In another aspect, the IL-6 antibodies or antigen binding portionsthereof may be deimmunized to reduce their immunogenicity using thetechniques described in, e.g., PCT Publication Nos.: WO98/52976 andWO00/34317 (incorporated herein by reference).

Derivatized and Labeled Antibodies

An IL-6 antibody or antigen-binding portion can be derivatized or linkedto another molecule (e.g., another peptide or protein). In general, theantibodies or antigen-binding portion thereof are derivatized such thatthe IL-6 binding is not affected adversely by the derivatization orlabeling. Accordingly, the antibodies and antigen-binding portions areintended to include both intact and modified forms of the human IL-6antibodies described herein. For example, an antibody or antigen-bindingportion can be functionally linked (by chemical coupling, geneticfusion, noncovalent association or otherwise) to one or more othermolecular entities, such as another antibody (e.g., a bispecificantibody or a diabody), a detection agent, a label, a cytotoxic agent, apharmaceutical agent, and/or a protein or peptide that can mediateassociation of the antibody or antigen-binding portion with anothermolecule (such as a streptavidin core region or a polyhistidine tag).

One type of derivatized antibody is produced by crosslinking two or moreantibodies (of the same type or of different types, e.g., to createbispecific antibodies). Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkersare available from Pierce Chemical Company, Rockford, Ill.

Another type of derivatized antibody is a labeled antibody. Usefuldetection agents with which an antibody or antigen-binding portion maybe derivatized include fluorescent compounds, including, for example,fluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-naphthalene-sulfonyl chloride, phycoerythrin,lanthanide phosphors. An antibody can also be labeled with enzymes thatare useful for detection, such as, for example, horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase, or glucose oxidase.When an antibody is labeled with a detectable enzyme, it is detected byadding additional reagents that the enzyme uses to produce a reactionproduct that can be discerned. For example, when the agent horseradishperoxidase is present the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody can also be labeled with biotin, and detectedthrough indirect measurement of avidin or streptavidin binding. Anantibody can also be labeled with a predetermined polypeptide epitoperecognized by a secondary reporter (e.g., leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags). In some cases, labels are attached by spacer arms of variouslengths to reduce potential steric hindrance. An IL-6 antibody can alsobe derivatized with a chemical group such as polyethylene glycol (PEG),a methyl or ethyl group, or a carbohydrate group. These groups areuseful to improve the biological characteristics of the antibody, e.g.,to increase serum half-life.

The specific Examples set forth herein are intended to illustrateparticular aspects of the disclosure and are not intended to limit thescope of the claims.

Example 1 Generation of Hybridomas Producing Anti-IL-6 Antibody

Exemplary antibodies in accordance with the disclosure were prepared,selected, and assayed as follows:

Mouse Strains

Fully human monoclonal antibodies to human IL-6 were prepared usinghuman Ig transgenic mouse strains HCo7 and HCo12, as well as the humantranschromosomal/transgenic strain, KM (Medarex, Inc.). These strainsall express fully human antibodies that are indistinguishable fromantibodies isolated from humans.

In all three strains, both the endogenous mouse kappa light chain geneand the endogenous mouse heavy chain gene have been homozygouslydisrupted as described in Chen et al. (1993) EMBO J. 12:811-820 and inExample 1 of PCT Publication WO 01/09187, respectively. Moreover, allthree carry a human kappa light chain transgene, KCo5, as described inFishwild et al. (1996) Nature Biotechnology 14:845-851. In contrast, thethree strains are distinct with respect to their human heavy chaingenes. The HCo7 strain carries the HCo7 human heavy chain transgene asdescribed in U.S. Pat. Nos. 5,545,806, 5,625,825, and 5,545,807; theHCo12 strain carries the HCo12 human heavy chain transgene as describedin Example 2 of PCT Publication WO 01/09187; and the KM strain carries ahuman mini-chromosome as described in Ishida et al., (2002), Cloning andStem Cells, 4: 91-102.

Immunization with IL-6 Antigen and Selection of HuMab Mice ProducingAnti-IL-6 Monoclonal Antibodies

General immunization schemes for HuMab mice are described in Lonberg etal (1994) Nature 368(6474): 856-859; Fishwild et al. (1996) NatureBiotechnology 14: 845-851 and PCT Publication WO 98/24884. In thepresent case, a total of 81 HuMab mice of the HCo7, HCo12 and KM strainswere immunized beginning at 6-16 weeks of age with 5-25 μgs of purifiedhuman IL-6 in Ribi adjuvant. Human IL-6 was isolated from a human bonemarrow-derived stromal cell, HS-5 (ATCC CRL-11882, Roecklein B. A. &Torok-Storb B., Blood 85: 997-1005, 1995), which endogenously secretesIL-6 into the media. Human IL-6 was purified from IL-6 expressing HS-5media, which was concentrated by ultrafiltration followed by a QSepharose anion exchange chromatography step and an affinitychromatography step using a mouse anti-hIL-6 MAb (R&D Systems, Catalognumber MAB2061, clone 1936). The purified human IL-6 had a purity ofabout 90% by SDS-PAGE. The major bands were excised from the SDS-PAGEgel, digested with trypsin and the extracted gel purified trypticpeptides were analyzed via MALDI/MS on the 4700 TOF/TOF ProteomicsAnalyzer and were confirmed to be human IL-6. Alternatively, one can userecombinant human IL-6 from commercial sources (for example, RecombinantHuman IL-6, catalog number 206-IL-6/CF. R&D System Inc. 614 MckinleyPlace NE, Minneapolis, Minn. 55413). Administration was via injectionintra-peritoneally, subcutaneously or into the footpad at 3-14 dayintervals, up to a total of 8 immunizations. Immune response wasmonitored via ELISA screening, as described below.

Selection of HuMab Mice Producing Anti-IL-6 Antibodies

Blood from the transgenic mice described above was obtained viaretro-orbital bleeds and analyzed by ELISA for specific binding topurified human IL-6 recombinant protein, as described by Fishwild et al.(1996), Nature Biotechnology 14: 845-851.

Briefly, microtiter plates were coated using 50 μl/well of a purifiedrecombinant IL-6 solution containing 1 μg/ml in PBS, and incubatedovernight at 4° C. The wells were then blocked using 200 μl/well of 5%chicken serum in PBS/Tween (0.05%). Dilutions of plasma fromIL-6-immunized mice were added to each well and incubated for 1 hour atambient temperature. The plates were washed with PBS/Tween and thenincubated with a goat-anti-human IgG Fc polyclonal antibody conjugatedwith horseradish peroxidase (HRP) for 1 hour at room temperature. Afterwashing, the plates were developed with ABTS substrate (Moss Inc,product #: ABTS-1000 mg/ml) and analyzed by spectrophotometer at OD415-495. Mice that developed the highest titers of anti-IL-6 antibodies(22 animals total) were used for fusions.

Generation of Hybridomas Producing Human Monoclonal Antibodies to IL-6:

The mice selected above were boosted intravenously with IL-6 at 3 daysand then again at 2 days prior to sacrifice and removal of the spleenand/or lymph nodes. A total of 17 fusions were performed.

The mouse splenocytes and/or lymph node lymphocytes isolated fromimmunized HuMab or KM mice, were fused to SP2/0 non-secreting mousemyeloma cells (ATCC, CRL-1581, ATCC American Type Culture Collection,1080 University Boulevard, Manassas, Va. 20110-2209 USA) usingelectrofusion (E-fusion, Cyto Pulse™ technology, Cyto Pulse™ Sciences,Inc., Glen Burnie, Md.), according to standard ormanufacturer-recommended protocols.

Briefly, single cell suspensions of splenocytes and/or lymph nodelymphocytes from immunized mice were prepared and then combined with anequal number of Sp2/0 non-secreting mouse myeloma cells; E-fusion wasthen performed.

The cells were then plated at 2×10⁴ cells/well in flat bottom microtiterplate, and incubated for 10-14 days in selective medium containing 10%fetal bovine serum, 10% P388D1 (ATCC, CRL-TIB-63) conditioned medium,3-5% (IGEN) in DMEM (Mediatech, Herndon, Va., Cat. No. CRL 10013, withhigh glucose, L-glutamine and sodium pyruvate), 5 mM HEPES, 0.055 mM2-mercaptoethanol, 50 mg/ml gentamycin and 1×HAT (Sigma, Cat. No.CRL-P-7185).

After 1-2 weeks, cells were cultured in medium in which the HAT wasreplaced with HT. Approximately 10-14 days after cell platingsupernatants from individual wells were screened for the presence ofhuman gamma, kappa antibodies. The supernatants which scored positivefor human gamma, kappa were then screened by ELISA (using the protocoldescribed above) for human anti-IL-6 monoclonal IgG antibodies. Theantibody-secreting hybridomas were transferred to 24 well plates,screened again and, if confirmed positive for human anti-IL-6 IgGmonoclonal antibodies, were subcloned at least twice by limitingdilution. The stable subclones were then cultured in vitro to generatesmall amounts of antibody in tissue culture medium for furthercharacterization.

Example 2 Sequencing of IL-6 Antibodies

Full-length anti-IL-6 antibodies were cloned and sequence verified fromhybridomas as follows: Poly(A)⁺ mRNA was isolated using an RNeasy MiniKit (Qiagen) and cDNA synthesized from the mRNA with the AdvantageRT-for-PCR kit (BD Biosciences) using oligo(dT) priming. The oligo(dT)primed cDNA for clone 9C8 was amplified using degenerate primers listedin Table 1 respectively.

TABLE 1 Degenerate primers (5′ to 3′) for 9C8 VH4_5UTR_FCTTTCTGAGASTCMTGGAKCTCMTG SEQ ID NO: 49 G_3UTR_R TACGTGCCAAGCATCCTCGCSEQ ID NO: 50 VK1a_5UTR_F GSARTCAGWCYCWVYCAGGACACAGC SEQ ID NO: 51K_3UTR_F AGGCTGGAACTGAGGAGCAGGTG SEQ ID NO: 52

Amplification was achieved using the High Fidelity Polymerase (Roche)and a PTC-200 DNA Engine (MJ Research) with cycling as follows: 2′@95°C.; 25×(20″@95° C., 30″@52° C., 2′@72° C.); 10′@72° C. PCR ampliconswere cloned into the pCR2.1 TOPO and transformed into TOP10 chemicallycompetent cells (Invitrogen) using the standard protocol. Clones weresequence verified using Grills 16^(th) BDTv3.1/dGTP chemistry (AppliedBiosystems Inc) and a 3730xl DNA Analyzer (Applied Biosystems Inc). Allsequences were analyzed by alignments to the ‘V BASE sequence directory’(Tomlinson, et al, J. Mol. Biol., 227, 776-798 (1992); Hum. Mol. Genet.,3, 853-860 (1994); EMBO J., 14, 4628-4638 (1995). The germline genesegment usages of exemplary anti IL-6 antibodies are listed in Table 2.

TABLE 2 Heavy chain Light chain Clone V_(H) D J_(H) V_(K) J_(K) Subtype9C8 4-34 — 3b L15 JK1 IgG1 22B5 4-34 — 3b L15 JK1 IgG1Full-Length Sequences of the ANTI-IL-6 Antibody Derived from Hybridomas9C8

DNA sequence of 9C8 heavy chain from hybridoma cells (variabledomain in uppercase) SEQ ID NO: 1CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTATCTATGGTGGGTCCTTCAGGGAGTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCTTTCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCAACATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGACCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGAGGAATTAGATGATTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaaDerived protein sequence (by translation) of 9C8 heavy chainfrom hybridoma cells (variable domain in uppercase) SEQ ID NO: 3QVQLQQWGAGLLKPSETLSLTCAIYGGSFREYYWSWIRQPPGKGLEWIGEIFHSGSTNYNPSLKSRVNISVDTSKNQFSLKLTSVTAADTAVYYCAREELDDFDIWGQGTMVTVSSastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkrvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwingkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkDNA sequence of 9C8 light chain from hybridoma cells (variabledomain in uppercase) SEQ ID NO: 12GACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGTAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAAAAGTTACCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAcgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgtDerived protein sequence (by translation) of 9C8 light chainfrom hybridoma cells (variable domain in uppercase) SEQ ID NO: 14DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYKSYPRTFGQGTKVEIKrtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgecANTI-IL-6 Antibodies Variable Domains were Cloned into ExpressionVectors as Follows:

The variable domains were amplified from pCR2.1 cloned cDNA usingprimers listed in Table 3. Amplification was achieved using the PfxPlatinum polymerase (Invitrogen) and a PTC-200 DNA Engine (MJ Research)with cycling as follows: 2′@94° C.; 20×(30″@94° C., 45″@55° C., 1′@68°C.); 5′@68° C. The variable domains were then cloned into expressionvectors containing constant domains of the appropriate isotype. Theseclones were sequence verified using Grills 16^(th) BDTv3.1/dGTPchemistry (Applied Biosystems Inc) and a 3730xl DNA Analyzer (AppliedBiosystems Inc).

TABLE 3 Variable domain primers (5′ to 3′) for 9C8 H4_34ttacagtGCGCGCACTCCCAGGTGCAGCTACAGCAGTGG SEQ ID NO: 53 K_O12ttacagtGTGCACTCCGACATCCAGATGACCCAGTCTCC SEQ ID NO: 54 G1/2_ch1GAAGACCGATGGGCCCTTGG (Apal)_R SEQ ID NO: 55 JK1_RtatattccttaattaagttattctactcacGTTT GATTTCCACCTTGGTCCCT SEQ ID NO: 56

Full-Length Sequences of Recombinant ANTI-IL-6 Antibodies 9C8

DNA sequence of recombinant 9C8 lgG2 heavy chain (variabledomain in uppercase) SEQ ID NO: 23CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTATCTATGGTGGGTCCTTCAGGGAGTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCTTTCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCAACATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGACCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGAGGAATTAGATGATTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAgcctccaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtagtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgtcgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctccgggtaaaDerived protein sequence (by translation) of recombinant 9C8lgG2 heavy chain (variable domain in uppercase) SEQ ID NO: 24QVQLQQWGAGLLKPSETLSLTCAIYGGSFREYYWSWIRQPPGKGLEWIGEIFHSGSTNYNPSLKSRVNISVDTSKNQFSLKLTSVTAADTAVYYCAREELDDFDIWGQGTMVTVSSastkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdktverkccvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqk slslspgkDNA sequence of recombinant 9C8 Kappa light chain (variabledomain in uppercase) SEQ ID NO: 25GACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGTAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAAAAGTTACCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAcgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgtDerived protein sequence (by translation) of recombinant 9C8Kappa light chain (variable domain in uppercase) SEQ ID NO: 26DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYKSYPRTFGQGTKVEIKrtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgec

Example 3 Mutagenesis of Anti-IL-6 Antibody

Amino acid substitution variants of the anti-IL-6 antibody 9C8 in theheavy chain variable domain were made at positions 24 (124V), 30 (R30S),31 (E31 G), 52 (F52N), 68 (N68T), 83 (T83S) either singly or incombination both in the context of an IgG1 or an IgG2 format. Amino acidsubstitution variants of the anti-IL-6 antibody 9C8 in the light chainvariable domain were made at 92 (K92N) both as an IgG1 or an IgG2format. Antibodies having both a heavy chain variable domain and a lightchain variable domain variant were made. Some of the various mutationcombinations are shown in Table 6a and 6b. Mutagenesis, in the V_(H)(124V), V_(H) (E31 G), V_(H) (N68T), and V_(H) (T83S) regions of clone9C8, was conducted with the primers listed in Table 4 (sense strandsshown; targeted residue shown in bold) and the QuickChange kit(Stratagene) according to the manufacturer's instructions. The mutatedvariants were sequence verified and cloned into expression vectors bystandard procedures.

TABLE 4   Mutagenic primers (5′ to 3′) for 9C8 9C8_H_I24VCCTCACCTGCGCTGTCTATGGTGGGTCC SEQ ID NO: 57 9C8_H_E31GGGGTCCTTCAGGGGGTACTACTGGAGCTG SEQ ID NO: 58 9C8_H_N68TCCTCAAGAGTCGAGTCACCATATCAGTAGACACG SEQ ID NO: 59 9C8_H_T83SCTCCCTGAAGCTGAGCTCTGTGACCGCC SEQ ID NO: 60

DNA sequence of recombinant 9C8 lgG2 heavy chain (N68T, T83S)heavy chain SEQ ID NO: 27CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTATCTATGGTGGGTCCTTCAGGGAGTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCTTTCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGAGGAATTAGATGATTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCAgcctccaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtagtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgtcgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaaDerived protein sequence (by translation) of recombinant 9C8lgG2 heavy chain (N68T, T83S) heavy chain SEQ ID NO: 28QVQLQQWGAGLLKPSETLSLTCAIYGGSFREYYWSWIRQPPGKGLEWIGEIFHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREELDDFDIWGQGTMVTVSSastkgpsvfplapcsrstsestaalgolvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdktverkccvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqk slslspgkDNA sequence of recombinant 9C8 lgG1 heavy chain (N68T, T83S)heavy chain SEQ ID NO: 31CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTATCTATGGTGGGTCCTTCAGGGAGTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCTTTCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGAGGAATTAGATGATTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCAgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtagtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaaProtein sequence of recombinant 9C8 lgG1 heavy chain (N68T, T83S)heavy chain SEQ ID NO: 32QVQLQQWGAGLLKPSETLSLTCAIYGGSFREYYWSWIRQPPGKGLEWIGEIFHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREELDDFDIWGQGTMVTVSSastkgpsvfplapsskstsggtaalgolvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwingkeykckvsnkalpapiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkVariable domain DNA sequence of recombinant 9C8 (E31G, N68T,T83S) heavy chain SEQ ID NO: 33CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTATCTATGGTGGGTCCTTCAGGGGGTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCTTTCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGAGGAATTAGATGATTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCAVariable domain translated protein sequence of recombinant 9C8(E31G, N68T, T83S) heavy chain SEQ ID NO: 34QVQLQQWGAGLLKPSETLSLTCAIYGGSFRGYYWSWIRQPPGKGLEWIGEIFHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREELDDFDIWGQGTMVTVSSVariable domain DNA sequence of recombinant 9C8 (124V, N68T,T83S) heavy chain SEQ ID NO: 36CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGGGAGTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCTTTCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGAGGAATTAGATGATTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCAVariable domain translated protein sequence of recombinant 9C8(124V, N68T, T83S) heavy chain SEQ ID NO: 37QVQLQQWGAGLLKPSETLSLTCAVYGGSFREYYWSWIRQPPGKGLEWIGEIFHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREELDDFDIWGQGTMVTVSS

The CMV promoter containing expression vectors were transfected in 293Freestyle (Invitrogen) cells according to the vendor's protocol.Supernatants from these cells were collected by centrifugation andpurified by standard Protein-A affinity chromatography to isolaterecombinant immunoglobulins. These proteins were then characterized bySDS-PAGE, light scatter, and spectrophotometrically.

The heavy and light chains of the anti-IL-6 antibodies indicated inTable 5 were deposited under terms in accordance with the BudapestTreaty with the American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. 20110-2209. The heavy & light chainshave been assigned the following accession numbers:

TABLE 5 Clone ATCC Patent Deposit Deposit Antibody DesignationDesignation Designation Date 9C8 heavy E. coli; pCR2.1 UC25510 PTA-8013Nov. 21, chain IgG1 TOPO 2006 9C8H(WT) 9C8 light chain E. coli; pCR2.1UC25511 PTA-8014 Nov. 21, IgG1 TOPO 2006 9C8L(WT) 9C8 N68T, E. coli;pCR2.1 UC25512 PTA-8015 Nov. 21, T83S IgG2 TOPO 2006 heavy chain9C8H(NT)hG2 9C8 N68T, E. coli; pCR2.1 UC25513 PTA-8016 Nov. 21, T83SIgG2 TOPO 2006 light chain 9C8L(wt)hk 9C8 E31G, E. coli; pCR2.1 UC25514PTA-8017 Nov. 21, N68T, T83S TOPO 2006 variable region 9C8H(ENT) 9C8I24V, E. coli; pCR2.1 UC25515 PTA-8018 Nov. 21, N68T, T83S TOPO 2006variable region 9C8H(INT) 9C8 N68T, E. coli; pCR2.1 UC25526 PTA-8019Nov. 21, T83S IgG1 TOPO 2006 heavy chain 9C8H(NT)hG1

Example 4 TF-1 Proliferation

Human cells, TF-1 cells, from American Type Culture Collection (ATCC)(Manassas, Va.), were obtained and maintained in RPMI-1640 mediumcontaining 10% heat inactivated fetal bovine serum (FBS) (Invitrogen,Carlsbad, Ca.), with 2 ng/ml recombinant human GM-CSF. TF-1 cells weresplit to 1-2×10⁵ for next day use. Prior to plating, the cells werewashed three times with RPMI-1640, counted, and the volume adjusted withassay medium to yield 2×10⁵ cells/ml. To each well, 50 μl of the washedcells was added and incubated overnight at 37° C. with 5% CO₂. Allconditions were conducted in triplicate in 96-well tissue culturetreated plates (Corning, Corning, N.Y.). To each well, either 25 ng/mlor 2.5 ng/ml IL-6 in a volume of 25 μl and test or control antibodies atvarious concentrations in a volume of 25 μl in sodium phosphate buffer(10 mM sodium phosphate and 150 mM sodium chloride, pH 7.4) to a finalvolume of 100 μl was added. Antibodies were tested alone and with humanIL-6. The plates were incubated for 48 hours (hrs) at 37° C. with 5%CO₂. After 48 hours, 10 μl/well of 0.5 μCi ³H-thymidine (AmershamBiosciences, Piscataway, N.J.) was added and pulsed with the cells for 3hrs. To detect the amount of incorporated thymidine, the cells wereharvested onto pre-wet unifilter GF/C filterplates (Packard, Meriden,Ct.) and washed 10 times with water. The plates were allowed to dryovernight. Bottom seals were added to the filterplates. Next, 45 μlMicroscint 20 (Packard, Meriden, Ct.) per well was added. After a topseal was added, the plates were counted in a Trilux microbeta counter(Wallac, Norton, Ohio) and data is reported as CPM (counts per minute).Tables 6a and 6b show the IC50s in the TF-1 proliferation assay of the9C8 antibody and the various antibodies having amino acid substitutions.The results shown in Table 6a and Table 6b are from assays done on twoseparate occasions.

TABLE 6a Average IC50 Fold Hc Lc (μg/ml) Difference 9C8 9C8 0.0022 1.09C8 I24V 9C8 0.0071 3.3 9C8 R30S 9C8 0.0260 12.1 9C8 N68T 9C8 0.0028 1.39C8 T83S 9C8 0.0026 1.2 9C8 I24V, N68T, T83S 9C8 0.0145 6.7 9C8 I24V,R30S, N68T, T83S 9C8 0.2040 94.9 9C8 E31G 9C8 0.0051 2.4 9C8 F52N 9C81.3850 644.2 9C8 9C8 K92N 0.0120 5.6

TABLE 6b Average IC50 Fold Hc Lc (μg/ml) Difference 9C8 9C8 0.0031 1.49C8 N68T, T83S 9C8 0.0030 1.4 9C8 E31G, N68T, T83S 9C8 0.0083 3.9 9C8I24V, E31G, N68T, T83S 9C8 0.0528 24.5 9C8 N68T, T83S 9C8 9C8 0.0186 8.6K92N 9C8 E31G, N68T T83S 9C8 K92N 0.1015 47.2 9C8 I24V, E31G N68T, T83S9C8 K92N 0.9670 449.9 Hc = heavy chain, Lc = light chain

Example 5 C-Reactive Protein from LPS-Monkey Study

The in-vivo portion of this study was conducted by Charles RiverLaboratories Preclinical Services at their Worcester, Mass. TestFacility. Briefly, the study consisted of fifteen male cynomolgusmonkeys (five groups; 3 monkeys/group). On Day 1 animals in group 1received vehicle; animals in Groups 2 and 3 received the antibody 9C8N68T T83S IgG₁ at doses of 0.5 and 5 mg/kg, respectively; and animals inGroups 4 and 5 received 9C8 N68T T83S IgG₂ at doses of 0.5 and 5 mg/kg,respectively. All treatments were administered by IV bolus injection atdose volumes of 1 mL/kg each. Approximately 2 hours after treatment, allanimals were challenged with 10 μg/kg bacterial lipopolysaccharide (LPS)at a volume of 1 mL/kg by slow IV bolus injection. On Day 1 blood wascollected from a peripheral vessel at baseline (prior to treatment);immediately following treatment; 2 hours post-treatment (immediatelyprior to LPS administration); and at 30 minutes and 1, 2, 3, 4, 6, 8,and 22 hours after LPS challenge. Blood samples were also collected onDays 3, 4, 5, 6 and 7. Whole blood samples were processed for serum andserum samples were stored frozen. C-Reactive Protein (CRP) was measuredusing a human Vascular Injury Panel II Multi-Spot® Assay Kit from MesoScale Discovery (MSD®), Gaithersburg, Md. Assays were performed asoutlined in published kit instructions from MSD. FIG. 2 shows the totalserum CRP for the 9C8 N68T T83S IgG₂ antibody.

Example 6 pSTAT3 Assay by Flow Cytometry

In vitro assays were conducted in human whole blood and human peripheralblood mononuclear cells (PBMCs) stimulated with recombinant human IL-6(rhIL-6) to measure phosphorylated STAT3 (pSTAT3) levels in the presenceof anti-IL-6 antibodies.

Whole Blood Assay

Freshly collected heparinized human whole blood was incubated withanti-IL-6 antibody or vehicle control (final volume 300 μL in 15 mLpolypropylene tube) for 15 minutes at 37° C. Samples were stimulatedwith recombinant human (rh) IL-6 for a final concentration of 25 ng/mLand incubated for 10 minutes at 37° C. Red blood cells (RBCs) were thenlysed with 4 mL RBC Lysing Buffer (Sigma), and samples were mixed gentlyfor 10 minutes at 37° C. Samples were spun at 400×g for 5 min to pelletcells. Supernatants were removed. Two milliliters of wash buffer (4% BSAin PBS, Gibco) were added to each tube, and then samples were spun at400×g for 5 min to pellet cells. Supernatants were removed, and cellpellets were resuspended in 200 μL of preheated (37° C.)permeabilization buffer (2% formaldehyde in PBS, Polysciences, Inc.) andincubated at 37° C. for 10 minutes. Three milliliters of ice cold MeOHwas added to each sample to fix the cells. Samples were mixed and placedon ice for at least 30 min. Samples were spun (400×g for 5 min.) andsupernatants were removed. Pellets were washed once with wash buffer.Cell pellets were then stained with anti-phosphorylated STAT3 (Y705)antibody conjugated to Alexa Fluor 488 (BD Pharmingen) diluted in washbuffer, final volume 100 μL. Samples were incubated on ice for 30minutes then washed 2× in wash buffer. Final cell pellets wereresuspended in 400 μL IF Buffer (Hank's Buffered Saline Solution, 2%Fetal Calf Serum, 10 mM Hepes, 0.2% Sodium Azide, Gibco). A FACSCaliburinstrument using CellQuest software (BD Biosciences) was used to collectand analyze data. Table 7 shows the pSTAT3 levels in the presence ofanti-IL-6 antibodies as measured in human whole blood.

TABLE 7 IL-6 Conc WB IC50 Anti IL-6 Antibody ng/ml μg/ml Assay # 9C8 250.487 1 25 0.432 2 25 0.482 3 I24V, N68T, T83S 25 0.420 1 25 0.527 2 250.749 3 E31G, N68T, T83S 25 0.626 1 25 0.714 2 25 0.968 3 I24V, E31G,N68T, T83S 25 0.364 1 25 0.603 2 25 0.821 3

PBMC Assay

Peripheral blood mononuclear cells (PBMCs) were isolated from freshlycollected heparinized human whole blood using Accuspin System-Histopaque1077 columns according to manufacture's protocol (Sigma-Aldrich A7054)and placed in (0.1% penicillin streptomycin in Macrophage SFM Media,Gibco). Approximately 2×10⁶ PBMCs were incubated with anti-IL-6 antibodyor vehicle control (final vol. 300 μL) for 15 min. at 37° C. PBMCs werestimulated with rhIL-6 for a final concentration of 25 ng/mL andincubated for 10 min. at 37° C. Samples were spun at 400×g for 5 min topellet cells. Supernatants were removed. Two mL wash buffer (4% BSA inPBS) was added to each sample. Samples were spun at 400×g 5 min topellet cells. Supernatants were removed and cell pellets wereresuspended in 200 uL of preheated (37° C.) permeabilization buffer (2%formaldehyde in PBS, Polysciences, Inc.) and incubated at 37° C. for 10min. Three milliliters of ice cold MeOH was added to each sample to fixthe cells. Samples were mixed and kept on ice for at least 30 min.Samples were spun at 400×g for 5 min. and supernatants were removed.Cell pellets were washed once with wash buffer then stained and analyzedas described in the whole blood assay. Table 8 shows the pSTAT3 levelsin the presence of anti-IL-6 antibodies as measured in human peripheralblood mononuclear cells.

TABLE 8 IL-6 conc. PBMC IC50 Anti IL-6 Antibody ng/ml μg/ml 9C8 25 0.210R&D MAB2061 25 0.580

Example 7 Binding Affinity Preparation of BIAcore Chips

The anti-IL-6 antibody 9C8 N68T T83S IgG2 was immobilized onto BIAcoreCM5 chips (GE Biosciences—formerly BIAcore Inc, Piscataway, N.J.) byamine coupling to carboxymethylcellulose (CM) attached to the dextranmatrix of the chip as described by Löfås & Johnsson (J. Chem. Soc. Chem.Commun. (1990); 21:pp 1526-1528). The chip was pretreated with 1 M NaCland 50 mM NaOH prior to coupling. Amine coupling was accomplished bycombining EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and NHS(N-hydroxysuccinimide) and passing across the chip to activate the CMgroups. The ligand in 10 mM acetate buffer pH 5 was passed across thechip surface and covalently bound to the activated CM group. Theremaining active CM groups on the chip surface were quenched by passing1M ethanolamine pH 8.5 across the chip. The EDC, NHS, and ethanolaminewere obtained as part of the Amine Coupling kit obtained from GEBiosciences. Coupling was performed using the automated SurfacePreparation Wizard included with the BiaControl Software V3.2.

Determination of Binding Affinity

All SPR measurements were performed on a BIAcore 3000 instrument (GEBiosciences, Piscataway, N.J.). BIAcore Software-BIAcore 3000 ControlSoftware V3.2 was used for the operation and control of the BIAcore 3000instrument. BiaEvaluation Software V4.1 was used for the analysis of SPRdata from the BIAcore 3000 instrument and data was plotted using GraphPad Prism Software Version 5. The binding affinity of IL-6 to monoclonalantibodies (mAb) was measured in HBS-EP buffer (10 mM HEPES, 150 mMNaCl, 3.4 mM EDTA, 0.005% P20) at 25° C. The flow rate for the affinitystudy was 40 uL/minute to minimize mass transport effects (Myszka, D.G., et al., Biophysical chemistry. 64, 127-137, 1997). Human IgG_(K) wasused as the ligand for the construction of the reference channel of thechip. Analyte (rhIL-6; R&D Systems, Minneapolis, Minn., 206-IL) bindingto the immobilized ligand (9C8 N68T T83S IgG₂) was measured in duplicateand the concentration of the IL-6 ranged from 0 to 25 nM. Injection timefor IL-6 was 6 minutes and dissociation time was 25 minutes. The surfacewas regenerated between cycles by 10 mM glycine pH 1.7 for 30 seconds ata flow rate of 30 uL/min. The regeneration conditions were establishedto be optimal after a regeneration study (data not shown). Data wasanalyzed by using the Kinetics Wizard and the manual fitting programsthat are both included with the BiaEvaluation Software V4.1 using a 1:1Langmuir model (Karlson R & Fält A., J. Immunol. Methods. 200: pp121-133, 1997). The anti IL-6 antibody 9C8 N68T T83S IgG₂ was shown tohave k_(a)=9.95E+05 (Ms)⁻¹, a k_(d)=1.34E−04 s⁻¹, and a K_(D) ⁼1.35 E−10M or 135 pM.

SUMMARY OF SEQUENCE LISTING (sequences are amino acid sequences exceptthose indicated by ‘n.a.’ for nucleic acid) SEQ ID NO: SEQUENCE 1 heavy9C8 IgG1 n.a. 2 V_(H) 9C8 n.a. 3 heavy 9C8 IgG1 4 V_(H) 9C8 5 V_(H) CDR19C8 6 V_(H) CDR2 9C8 7 V_(H) CDR3 9C8 8 V_(H) FR1 9C8 9 V_(H) FR2 9C8 10V_(H) FR3 9C8 11 V_(H) FR4 9C8 12 light 9C8 IgG1 n.a. 13 V_(L) 9C8 n.a.14 light 9C8 IgG1 15 V_(L) 9C8 16 V_(L) CDR1 9C8 17 V_(L) CDR2 9C8 18V_(L) CDR3 9C8 19 V_(L) FR1 9C8 20 V_(L) FR2 9C8 21 V_(L) FR3 9C8 22V_(L) FR4 9C8 23 heavy 9C8 IgG2 n.a. 24 heavy 9C8 IgG2 25 light 9C8 IgG2n.a. 26 light 9C8 IgG2 27 heavy 9C8 N68T T83S IgG2 n.a. 28 heavy 9C8N68T T83S IgG2 29 V_(H) 9C8 N68T T83S 30 V_(H) FR3 9C8 N68T T83S 31heavy 9C8 N68T T83S IgG1 n.a. 32 heavy 9C8 N68T T83S IgG1 33 V_(H) 9C8E31G N68T T83S n.a. 34 V_(H) 9C8 E31G N68T T83S 35 V_(H) CDR1 9C8 E31GN68T T83S 36 V_(H) 9C8 I24V N68T T83S n.a. 37 V_(H) 9C8 I24V N68T T83S38 V_(H) FR1 9C8 I24V N68T T83S 39 V_(H) 22B5 n.a. 40 V_(H) 22B5 41V_(H) CDR3 22B5 42 V_(L) 22B5 n.a. 43 V_(L) 22B5 44 Consensus 1 CDR3 45Consensus 2 CDR3

1. An isolated human monoclonal antibody or antigen-binding portionthereof comprising a heavy chain variable (V_(H)) domain amino acidsequence comprising a CDR1, CDR2, and CDR3 region selected from thegroup consisting of: a) a V_(H) CDR1 as set forth in SEQ ID NO: 5, aV_(H) CDR2 as set forth in SEQ ID NO:6, and a V_(H) CDR3 as set forth inSEQ ID NO:7; b) a V_(H) CDR1 as set forth in SEQ ID NO: 35, a V_(H) CDR2as set forth in SEQ ID NO:6, and a V_(H) CDR3 as set forth in SEQ IDNO:7; c) a V_(H) CDR1 as set forth in SEQ ID NO: 5, a V_(H) CDR2 as setforth in SEQ ID NO:6, and a V_(H) CDR3 as set forth in SEQ ID NO:41; d)a V_(H) CDR1 as set forth in SEQ ID NO: 5, a V_(H) CDR2 as set forth inSEQ ID NO:6, and a V_(H) CDR3 as set forth in SEQ ID NO:44; e) a V_(H)CDR1 as set forth in SEQ ID NO: 5, a V_(H) CDR2 as set forth in SEQ IDNO:6, and a V_(H) CDR3 as set forth in SEQ ID NO:45; wherein saidantibody binds specifically to human IL-6.
 2. (canceled)
 3. An isolatedhuman antibody or antigen-binding portion thereof comprising a lightchain variable (V_(L)) domain amino acid sequence comprising a V_(L)CDR1 as set forth in SEQ ID NO:16, a V_(L) CDR2 as set forth in SEQ IDNO:17, and a V_(L)CDR3 as set forth in SEQ ID NO: 18; wherein saidantibody binds specifically to human IL-6. 4-9. (canceled)
 10. Anisolated human monoclonal antibody or antigen-binding portion thereofcomprising V_(L) and V_(H) domains at least 90% identical in amino acidsequence to the V_(L) and V_(H) domains, respectively, of antibody 9C8,9C8 N68T T83S, 9C8 E31G N68T T83S, 9C8 I24V N68T T83S, and 22B5; whereinsaid antibody binds specifically to human IL-6. 11-18. (canceled)
 19. Anisolated nucleic acid molecule comprising a nucleotide sequence thatencodes an antibody according to claim
 1. 20. A vector comprising thenucleic acid molecule according to claim 19, wherein the vectoroptionally comprises an expression control sequence operably linked tothe nucleic acid molecule.
 21. A host cell comprising the vectoraccording to claim
 20. 22. A host cell comprising the nucleic acidmolecule according to claim
 19. 23. A method for treating, preventing oralleviating the symptoms of a IL-6 mediated disorder in a subject inneed thereof, comprising administering to said subject an antibody orantigen-binding portion according to claim
 1. 24-28. (canceled)